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Journal of Bacteriology, September 2000, p. 5114-5120, Vol. 182, No. 18
Department of Medical Biochemistry and
Molecular Biology, Institute of Biomedicine, University of Turku,
FIN-20520 Turku, Finland
Received 27 March 2000/Accepted 18 June 2000
Bacteriophage Yersinia enterocolitica
is a Gram-negative species which contains several serotypes, some of
which are pathogenic to humans. The major pathogens in Europe, Canada,
Japan, and South Africa belong to serotypes O:3 and O:9, and those in
the United States belong to serotype O:8 (11). The main
reservoir in nature for Y. enterocolitica is pigs
(15), and human infections usually take place after
ingestion of contaminated foodstuffs.
A number of yersiniophages have been described, but only a few have
been characterized by electron microscopy and to our knowledge none
have been studied in detail. In our laboratory a number of Yersinia-specific bacteriophages have been isolated, all
originating from the raw incoming sewage of the Turku City sewage
treatment plant, and the phages have been used as genetic tools
(32). One of the phages, Culture conditions.
Bacterial strains, bacteriophages and
plasmids used in this study are listed in Table
1. Virulence plasmid-cured Y. enterocolitica serotype O:3 strain 6471/76-c (31) was
the usual host for propagation of phage
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Bacteriophage
YeO3-12, Specific for
Yersinia enterocolitica Serotype O:3, Is Related to
Coliphages T3 and T7
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
YeO3-12 is a lytic phage of Yersinia
enterocolitica serotype O:3. The phage receptor is the
lipopolysaccharide O chain of this serotype that consists of the rare
sugar 6-deoxy-L-altropyranose. A one-step growth curve of
YeO3-12 revealed eclipse and latent periods of 15 and 25 min,
respectively, with a burst size of about 120 PFU per infected cell. In
electron microscopy YeO3-12 virions showed pentagonal outlines,
indicating their icosahedral nature. The phage capsid was shown to be
composed of at least 10 structural proteins, of which a protein of 43 kDa was predominant. N-terminal sequences of three structural proteins
were determined, two of them showing strong homology to structural
proteins of coliphages T3 and T7. The phage genome was found to consist
of a double-stranded DNA molecule of 40 kb without cohesive ends. A
physical map of the phage DNA was constructed using five restriction
enzymes. The phage infection could be effectively neutralized using
serum from a rabbit immunized with whole YeO3-12 particles. The
antiserum also neutralized T3 infection, although not as efficiently as that of YeO3-12. YeO3-12 was found to share, in addition to the
N-terminal sequence homology, several common features with T3,
including morphology and nonsubjectibility to F exclusion. The evidence
conclusively indicated that YeO3-12 is the first close relative of
phage T3 to be described.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
YeO3-12, was isolated as
specific to Y. enterocolitica serotype O:3. The phage could
infect Escherichia coli C600 expressing the cloned O antigen
of Y. enterocolitica serotype O:3 and spontaneous phage-resistant Y. enterocolitica serotype O:3 strains were
missing the O antigen, indicating that the O antigen is the phage
receptor (4, 5). The serotype O:3 specificity makes the
phage YeO3-12 a potential biotechnological tool, and therefore we
have initiated its detailed characterization. Here we present the
biological and physical properties of the phage and evidence suggesting
that YeO3-12 is closely related to coliphages T3 and T7.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
YeO3-12. The YeO3-12 and
its host are available under accession no. HER 249 and 1249, respectively, at the Felix d'Herelle Reference Center for Bacterial
Viruses. Bacterial strains were grown in tryptone soya broth medium
(TSB; Oxoid), and incubations were done at room temperature (RT; 22 to
25°C) unless specified otherwise. E. coli strains were
grown in Luria broth (LB) at 37°C, and ampicillin (100 µg/ml) was
added when required. Solid medium was obtained by adding 2% (wt/vol)
agar to LB, and soft agar was obtained by adding 0.5% (wt/vol) agar to
TSB (7, 30).
TABLE 1.
Bacterial strains, bacteriophages, and plasmids used in
this study
Propagation and purification of phage YeO3-12.
Bacteriophage YeO3-12 (Table 1) was stored at 
70°C in TSB
supplemented with 7% dimethyl sulfoxide (DMSO). Large-scale
purification of YeO3-12 virions was done as described elsewhere
(30). Briefly, an overnight culture of Y. enterocolitica serotype O:3 strain 6471/76-c was diluted 10-fold
in TSB in a total volume of 1 liter divided into four 2-liter
Erlenmeyer flasks and infected with YeO3-12 at a multiplicity of
infection (MOI) of 1. The infected cultures were incubated at 25°C
with vigorous aeration (250 rpm), until, usually after 2.5 h, the
bacterial lysis took place. The lysed culture was treated with DNase I
(1.2 µg/ml; Roche Molecular Biochemicals) and RNase A (1 µg/ml;
Sigma Chemicals, St. Louis, Mo.) at RT for 30 min. Sodium chloride
(final concentration, 1 M) was added to the treated lysate and
incubated on ice for 1 h, and then the solution was centrifuged at
11,000 × g at 4°C for 20 min in a Sorvall GS-3
fixed-angle rotor to remove the precipitated bacterial debris. The
phage was recovered from the supernatant by precipitating with
polyethylene glycol (PEG) 8000 (10%, wt/vol; 
60 min; 0°C) and was
resuspended into SM buffer (30). The phage was further
purified by chloroform extraction and one to three rounds of
discontinuous glycerol density gradient ultracentrifugation at 35,000 rpm at 4°C for 4 h in a Sorvall TH-641 swing-out rotor. After
ultracentrifugation the phages were resuspended in SM buffer containing
8% sucrose to yield a typical concentration of ca. 1014
PFU/ml, as determined by phage titration (30).
Host range determination.
The host range of YeO3-12 was
determined by pipetting 20-µl droplets of serial dilutions of
concentrated phage stocks (up to 1014 PFU/ml) on lawns of
different bacterial strains prepared on LB plates. The formation of
plaques of lysis, i.e., the plating efficiency, was tested both at RT
and at 37°C.
Electron microscopy. Phage particles were negatively stained with 2% (wt/vol) phosphotungstic acid, pH 7. Prior to examination, the particles were sedimented at about 25,000 × g for 60 min in a Beckman (Palo Alto, Calif.) J2-21 centrifuge, using a JA-18.1 fixed-angle rotor. This was followed by two washes in 0.1 M ammonium acetate, pH 7.2. Stained particles were observed in a Philips EM 300 electron microscope operated at 60 kV. Magnification was monitored with catalase crystals (Worthington, Freehold, N.J.) (24). Dimensions were measured on photographic prints at a final magnification of ×297,000.
Density and fatty acid analysis. A PEG-precipitated bacteriophage suspension was loaded on a continuous CsCl density gradient and ultracentrifuged to equilibrium at 40,000 rpm (Sorvall TST 60.4) for 24 h at 4°C. After centrifugation, fractions were collected for phage titration and density measurement. For fatty acid analysis the phage particles were treated with sodium dodecyl sulfate (SDS) (0.5%) and proteinase K (0.2 mg/ml) for 70 min at 56°C, extracted, and converted into methylester derivatives which were analyzed by gas chromatography (18).
One-step growth curve. A mid-exponential-phase culture (10 ml) of Y. enterocolitica serotype O:3 strain 6471/76-c (optical density at 600 nm [OD600], 0.4 to 0.5) was harvested by centrifugation and resuspended in 0.25 volume of fresh TSB (ca. 109 CFU/ml). Phage was added at an MOI of 0.0005 and allowed to adsorb for 5 min at RT. The mixture was then centrifuged, pelleted cells were resuspended in 10 ml of TSB, and incubation was continued at RT. Samples were taken at 5-min intervals. The first set of samples was immediately diluted and plated for phage titration. A second set of samples was treated with 1% (vol/vol) chloroform to release intracellular phages in order to determine the eclipse period before phage titration (9).
Immune sera.
Immunization of rabbits with bacteriophage
YeO3-12 was performed as follows. Preimmunization serum samples were
collected from the rabbits prior to immunization. Three young rabbits
were immunized with purified phages (ca. 1013 PFU/rabbit)
in Freund's complete adjuvant (Difco 0638) in phosphate-buffered saline (PBS) by subcutaneous injection of four sites (0.25 ml each) on
the back of the rabbit. Booster immunizations were done by injecting
phages in Freund's incomplete adjuvant (Sigma F-5506) every 3rd week
for three times. Humoral immune responses were monitored by analyzing
serum samples by enzyme immunoassay (EIA) for specific antibodies on
the 5th and 8th weeks of immunization (see below). The rabbits were
killed and the blood was collected after 10 weeks of immunization. The
sera were separated from the blood after clotting and were stored
frozen at 20°C (or 70°C for longer periods).
EIA.
The wells of a 96-well microtiter plate (Nunc-Immuno
plate; MaxiSorp surface) were coated with heat-inactivated YeO3-12
(140 min at 80°C followed by 65 min at 95°C) in PBS (100 µl per
well containing about 1010 phage particles [ca. 1 µg])
overnight at RT. After three washes with PBS, the wells were blocked
with 150 µl of 5% skim milk powder (wt/vol) in water for 2 h at
37°C and then washed again three times with PBS. The rabbit sera were
diluted in 1% normal sheep serum (NSS) in PBS to obtain twofold
dilutions between 1:8,000 and 1:256,000, and 75 µl of the dilutions
was incubated in the wells for 1.5 h at 37°C, after which the
wells were washed three times with PBS. Then 75 µl of swine
horseradish peroxidase-conjugated immunoglobulin against rabbit
immunoglobulins (P217; Dako A/S, Colostrup, Denmark) diluted 1:1,000 in
1% NSS-PBS was added and incubated for 1 h at 37°C. The plates
were washed three times with PBS, and 75 µl of a substrate solution
(3 mg of 1,2-phenylenediamine/ml and 0.02%
H2O2 in citrate buffer [per liter, 4.97 g
of citric acid × H2O and 9.9 g of
Na2HPO4 × 2H2O]) was added
and incubated for 10 min at RT. The reactions were terminated by adding
125 µl of 1 M HCl to the wells. Optical absorbances were measured at
492 nm with a Labsystems Multiskan MCC/340 Photometer. Each sample was
analyzed in duplicate. For negative controls, wells with 1:8,000
diluted preimmune sera from the rabbits and wells without any rabbit
serum were included.
YeO3-12 particles, the rabbit serum was diluted 1:2,000 and
1:10,000 in 1% NSS-PBS, and 75 µl of the dilutions was incubated in
the wells for 1.5 h at 37°C. After a wash step, the antibodies
that had bound to the phage particles were detected as described above
with the same negative controls. Each sample was analyzed in triplicate.
SDS-polyacrylamide gel electrophoresis (PAGE) and
immunoblotting.
Samples of purified virions of YeO3-12 were
heated at 95°C for 15 min in SDS gel-loading buffer (30).
Electrophoresis was conducted at a constant current of 13 mA in
SDS-10% polyacrylamide gels using the Hoefer SE 600 device (Amersham
Pharmacia Biotech) as described by Laemmli (23). After
electrophoresis the gels were either stained with Coomassie brilliant
blue R-350 (Pharmacia) or blotted to a nitrocellulose membrane (BAS 83;
Schleicher & Schuell, Dassel, Germany) using the Trans-Blot Semi-Dry
Transfer Cell (Bio-Rad), according to the manufacturer's instructions. The membrane was blocked overnight at RT in 5% skim milk powder in
PBS. After a wash, it was incubated with the strongest rabbit serum
(diluted 1:20,000 in 5% skim milk powder in PBS) overnight at RT. The
membrane was washed and then incubated with 1:2,000 diluted P217 for
2 h at RT. After four washes, the bound peroxidase was detected
using the ECL Western blotting kit (RPN 2106; Amersham International
plc, Little Chalfont, England).
Affinity purification of antibodies against YeO3-12
proteins.
Antibodies monospecific for phage proteins were affinity
purified from the rabbit antiserum as described elsewhere
(29). SDS-PAGE samples of purified virions of YeO3-12
were electrophoresed using a 10% polyacrylamide separation gel and
then transferred to a nitrocellulose membrane. The membrane was stained
with Ponceau S (2%, wt/vol) for 5 min to visualize the protein bands,
and the membrane was excised into 27 horizontal strips. The strips were incubated overnight at RT in 5% skim milk powder in PBS to block nonspecific binding sites. After a wash step, each strip was incubated overnight at RT with 1 ml of the strongest rabbit serum diluted 1:50 in
5% skim milk powder in PBS. The strips were washed four times with
PBS. Specific anti-protein antibodies were eluted by treatment with
1,050 µl of 0.2 M HCl-glycine buffer (pH 2.2) at RT for 15 min. The
pH of the eluate was immediately neutralized by the addition of 450 µl of 1 M K2HPO4, and then it was dialyzed overnight at 4°C against PBS. The antibodies were stored at 4°C.
Neutralization.
The crude rabbit serum, heat-inactivated
(56°C for 30 min) rabbit serum, and affinity-purified antibodies were
tested for their abilities to neutralize YeO3-12, T3, and T7
infections. The crude rabbit serum was also preincubated 10 min at RT
with an excess of T3 (107 PFU) before being tested for its
ability to neutralize YeO3-12 infection. The sera were diluted in
PBS to obtain twofold dilutions, and 50 µl of the dilutions was
incubated with a constant amount (ca. 100 PFU) of different phages for
10 min at RT. Then 150 µl of an overnight culture of indicator
bacteria (Y. enterocolitica serotype O:3 strain
6471/76-c or IJ855) was added, the mixture was plated, and plaques
were counted.
N-terminal amino acid analysis. SDS-PAGE of purified phage particles was run in a Mini-PROTEAN II gel electrophoresis apparatus (Bio-Rad). The electrophoresis and subsequent electroblotting onto Problott (ABI Foster City, Calif.) were performed according to the work of Moos et al. (28). Amino-terminal amino acid sequencing of the immobilized protein was performed with an Applied Biosystems 477A protein sequencer (ABI, Ltd.) equipped with an on-line ABI 120A PTH (phenylthiohydantoin)-amino acid analyzer. Standard cycle parameters provided by the manufacturer were used. Protein similarity searches were done at the European Bioinformatics Institute site (http://www.ebi.ac.uk/fasta3) using the Fasta3 search with default parameters against the SwissProt All library.
DNA techniques.
Phage DNA was obtained from high-titer phage
preparations as described by Sambrook et al. (30). Plasmid
DNA was extracted from E. coli by alkaline lysis
according to the procedure of Birnboim and Doly (10). All
enzymatic treatments of DNA were performed as recommended by the
manufacturers. DNA electrophoresis was carried out in agarose gels
using standard TAE buffer (30). A physical map of the DNA of
YeO3-12 was determined using five restriction enzymes
(BclI, EcoRV, PmlI, SnaBI,
and XbaI; New England Biolabs, Beverly, Mass.) by analysis
of single and double digests. The existence of cohesive ends was
assayed by comparing the restriction patterns of phage DNA with and
without prior treatment with T4 DNA ligase. Aliquots of phage DNA were
heated at 70°C for 10 min in order to melt the cohesive ends and then
were either slowly cooled down to RT or immediately placed on ice.
After ligation and digestion with DraI or StuI,
the samples were heated again at 70°C and then kept on ice until
loading in an agarose gel (19). Lambda DNA was used as a
positive control for cohesive ends.
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Host range of YeO3-12.
Previous work established that the
YeO3-12 receptor is a homopolymer of
6-deoxy-L-altropyranose, the O antigen of Y. enterocolitica serotype O:3 (4). The host range of
YeO3-12 was assayed using almost 300 strains belonging to eight
Yersinia species (Table 2). A
large number of Y. enterocolitica serotype O:3 strains were
included in order to test O:3 strains isolated from different origins.
The results (Table 2) showed that YeO3-12 could form plaques only on
Y. enterocolitica serotypes O:1, O:2, and O:3, i.e.,
serotypes with an O antigen known to contain
6-deoxy-L-altropyranose. No difference in sensitivity was
found between Y. enterocolitica serotype O:3 strains of
human and animal origin. Also, Yersinia frederiksenii
serotype O:3 and Yersinia mollaretii serotype O:3 were found
to be phage sensitive. No plaques were produced on strains of serotypes
of Y. enterocolitica or of other Yersinia species
where 6-deoxy-L-altropyranose is not present in the O antigen. Similar phage propagation in sensitive strains was observed at
RT and 37°C.
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YeO3-12. To find out whether this was due to
absence of the biosynthetic genes of the O:3 O antigen, the
Y. kristensenii genomic DNA was isolated and used as a
template in PCR. As a positive control, template DNA from
Y. enterocolitica O:3 was used. Two sets of primers
from the Y. enterocolitica O:3 O-antigen gene cluster
(accession no. Z18920) were used. Identical PCR products were
obtained from both species (data not shown). This suggested that
in the Y. kristensenii O:3 strain studied, the O-antigen expression was somehow inactivated or its surface exposure was blocked.
Morphology and physical properties of YeO3-12 particles.
Electron microscopy of phosphotungstic acid-stained YeO3-12
virions (Fig. 1) revealed particles with
approximate dimensions of 57 nm for the head and 15 by 8 nm for the
tail. Extended tail fibers were not seen. Normal capsids sometimes
showed pentagonal outlines, indicating their icosahedral nature. Based
on its morphology, YeO3-12 belongs to the family
Podoviridae (25) and to type C in Bradley's
classification (12); furthermore, it resembles a typical
member of the T7 group (H.-W. Ackermann, personal communication) (1). Other Y. enterocolitica phages
characterized to date by electron microscopy have been of type A in
Bradley's classification (22) or have been classified into
the families Myoviridae or Podoviridae
(2).
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YeO3-12 lost infectivity very rapidly when
CsCl was present. Fatty acid analysis failed to reveal any host-derived
fatty acids from the phage particle.
Size and structure of the YeO3-12 genome.
The phage genome
was shown to consist of double-stranded DNA after its digestion with
different restriction endonucleases (Fig.
2A). The size of the full-length phage
genome was estimated to be about 40 kb, i.e., close to that of
coliphages T3 and T7 (8, 17). Comparison of restriction
patterns of unligated and ligated phage DNA showed similar DNA fragment
patterns, indicating that YeO3-12 DNA does not have cohesive ends
(data not shown). A restriction map of the YeO3-12 DNA was
determined by analysis of single and double digests with restriction
enzymes BclI, EcoRV, PmlI,
SnaBI, and XbaI (Fig. 2).
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YeO3-12 did not clearly
indicate whether YeO3-12 is more closely related to T7 or to T3
(data not shown).
One-step growth curve.
The one-step growth curve of
YeO3-12 propagated on Y. enterocolitica
serotype O:3 strain 6471/76-c was determined and is shown in Fig.
3. Eclipse and latent periods of 15 and
25 min, respectively, were observed, followed by a short growth period of 10 min; the burst size was 100 to 140 PFU per infected cell. These
values fit into the range observed with T7 group phages.
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) and
in untreated cultures (
) at different time points. Each data point
is a mean from three experiments.
T7 group features of YeO3-12.
It is known that most members
of the T7 phage group abortively infect the host strain Shigella
sonnei D2 371-48, displaying the same pattern of
breakdown of newly synthesized phage DNA (21), while T3
plates normally on this host. S. sonnei D2
was transformed with pAY100, which encodes the YeO3-12
receptor. YeO3-12 plated normally on this host (data not shown). In
addition to this, T7 and most of its relatives, T3 being the exception,
fail to grow in cells harboring the F plasmid (26). Plating
assays of YeO3-12 on isogenic female and male hosts, IJ511
(F
) and IJ512 (F+), transformed with pAY100,
showed that YeO3-12 plated on IJ512/pAY100 (F+)
with essentially normal efficiency, and the plaques appeared normal in every way (data not shown).
YeO3-12, we looked
at the plating efficiencies of YeO3-12 on E. coli
RNA polymerase rpoC319 and rpoC320
mutants, again transformed with pAY100. T7+ does not form
plaques on rpoC319 cells but plates with near-normal efficiency on rpoC320 cells (13), whereas
T3+ productively infects rpoC319 cells
(14), suggesting that the T3 gp2 (gene product) interaction
with E. coli RNA polymerase differs from that of T7 gp2. In
line with the T3-like F plasmid results above, YeO3-12 plated
normally on the rpoC319/pAY100 cells (data not shown).
A very common type of variation encountered among different T7 group
phage strains is a deletion of some portion of the gene 0.7 region, and
plating efficiency on rpoC320 cells gives a good preliminary
indication whether a given phage strain carries a deletion
(34). T3 and T7 phages defective in the 0.7 gene, which codes for a protein kinase that is involved in host shutoff, are unable
to plate on rpoC320 cells (13); however,
rpoC320 is known to be less restrictive to 0.7 mutants of T3
than it is to 0.7 mutants of T7 (Ian Molineux, personal communication).
Furthermore, it is known that 0.7 deletion mutants grow better
(in the laboratory) than wild-type phages and that gene 0.7 is important for growth in poor media and at elevated temperatures
(20, 27). As we had serendipitously encountered a
faster-growing mutant of YeO3-12, we wondered whether this
mutant was defective in the 0.7 gene. The YeO3-12 mutant
(designated 
PK) showed no differences when compared to
YeO3-12 in plating efficiencies on rpoC319 and
rpoC320 cells (data not shown). YeO3-12 and PK
DNA were digested with the HpaI and EcoRV
restriction enzymes. Evident in the HpaI digestion was a ca.
0.7-kb difference in one of the fragments, indicating a
putative 1.75% deletion, and in the EcoRV digestion one of
the restriction sites was missing, and the 2.7- and 8.2-kb
fragments (see Fig. 2) were joined together, giving a ca. 10.2-kb
fragment (data not shown), placing the deletion close to one end of the phage genome. Nucleotide sequence analysis of the phages (unpublished data) confirms that PK indeed is a gene 0.7 deletion derivative of
YeO3-12, as suggested by the above results.
Antiserum specific for YeO3-12 particles.
All three
of the immunized rabbits developed good humoral immune responses
against YeO3-12 particles. In EIA analysis using phage
particles as the antigen, the mean absorbance values of the preimmune
rabbit sera at a 1:8,000 dilution were <0.1. The mean absorbance
values of the three rabbit sera at a 1:64,000 dilution were 0.211, 0.260, and 0.463, respectively, when heat-inactivated YeO3-12
particles were used as the antigen. The absorbance value of the
strongest serum at a dilution of 1:256,000 was still significantly higher than the background (0.208), and therefore it was used in
later experiments.
YeO3-12 particles were used as the antigen, and
the mean absorbance values of two times background were considered
significant. The sensitivity of 50 ng of antigen (phage in our case) is
very typical for an assay of this type (35).
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Analysis of the phage structural proteins.
SDS-PAGE of the
virion proteins (Fig. 5) revealed one
predominant polypeptide with a molecular mass of about 43 kDa (p43), which most likely is the major capsid protein (see the discussion of
N-terminal sequences below). From extensively purified phage particles
(four to five rounds of ultracentrifugation), at least 10 other
polypeptide bands ranging from ca. 15 to >200 kDa could also be
detected by Coomassie blue staining. Immunogenic phage proteins were
identified using rabbit antiserum specific for YeO3-12 (Fig. 5). The
immunostained protein bands were essentially identical to those seen in
the Coomassie-stained gel (Fig. 5), although some qualitative
differences were evident: p60, p100, and p200 were overrepresented in
the immunoblot. Also, some low-molecular-weight proteins gave
relatively stronger signals in the immunoblot than in
Coomassie staining. The tail fiber protein of T7 (gp17;
Mr, 61,441) forms a trimer, with an average mass
of 166 kDa for the trimer (33). It is possible that p60
could be monomeric gp17 of YeO3-12, and subsequently p200 could be a
gp17 trimer of YeO3-12.
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N-terminal amino acid analysis of phage proteins.
N-terminal
sequences of the most abundant proteins were determined (Table
3). This analysis indicated that the p43
N terminus was highly similar to that of the major capsid protein 10A
(gp10A) of phages T3 and T7 (Table 3). The N terminus of p53 was
identical to that of p43. This indicated that, as in T3 and T7, a
translational frameshift (1) may take place close to the end of the
p43 gene of YeO3-12, giving rise to a minor capsid protein
(16). The N-terminal sequence of p88 revealed no significant
similarities in the databases, indicating that it is a novel protein.
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Inhibition and neutralization of YeO3-12 infection.
To
characterize the host specificity mechanisms of phage YeO3-12,
inhibition and neutralization tests were performed. The phage infection
could be inhibited with purified lipopolysaccharide (LPS) (10 to 100 µg/ml) or with glycerol (1%). The effect of glycerol was noticed by
chance after purification of the phage by discontinuous glycerol
density gradients. Inhibition by glycerol was reversible when it was
diluted below 1%. The mechanism of inhibition by glycerol is not
known, but we assume that glycerol in high concentrations can
occupy the 6-deoxy-L-altropyranose binding site of the
phage adsorption complex. In addition, we found that YeO3-12 did not degrade the Y. enterocolitica O:3 O antigen when LPS samples
from bacteria exposed to the phage were compared to samples from intact bacteria (data not shown).
YeO3-12 by the strongest rabbit antiserum was
efficient; at a final dilution of 1:10,000, the antiserum inhibited
50% of the ca. 150 PFU of YeO3-12 (Fig.
6). At a final dilution of 1:20, up to
5 × 106 PFU of YeO3-12 was totally neutralized,
showing the high capacity of the antiserum. Heat treatment of the
antiserum at 56°C for 30 min did not affect its efficiency (data not
shown), indicating that activation of serum complement did not play a
role in neutralization. T7 was not inhibited by the antiserum, in
contrast to T3, which was totally neutralized by a 1:2 final dilution
and 50% neutralized by a 1:600 final dilution of the antiserum when
ca. 100 PFU of T3 particles was used in the assay. On the other hand,
T3 is inactivated by anti-T7 serum at about 10% of the homologous rate
(3).
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YeO3-12. Only
monospecific anti-p43 antibodies could neutralize YeO3-12 with about
95% efficiency; the other monospecific antibodies showed no
neutralizing activity. The latter finding could be due to one or more
of several different factors: (i) the only anti-p43 antibodies are
neutralizing, (ii) blotted adsorptive proteins have the wrong
conformation and the neutralizing antibodies do not bind to them,
and/or (iii) the amount of neutralizing antibodies obtained by the
strip affinity purification method was too small. In line with the last
point was the fact that anti-p53 antibodies did not show neutralizing
activity, but p53 was immunostained when anti-p43 antibodies were used
as the primary antibody in immunoblotting (data not shown). The
monospecific anti-p43 antibodies were also able to neutralize T3 with
about 50% efficiency (data not shown). Further work is needed to
elucidate the mechanism behind the neutralizing activity of the
anti-p43 antibodies.
Conclusions.
In electron microscopy YeO3-12 virions showed
pentagonal outlines, indicating their icosahedral nature, and thus
YeO3-12 was classified as a typical member of the T7 group. In line
with the morphology results, YeO3-12 showed T7 group features when plating was tested on an F-plasmid-containing host. On E. coli RNA polymerase mutants, YeO3-12 plated like T3. N-terminal
sequences of three structural proteins were determined, and two of them showed strong homology to structural proteins of coliphages T3 and T7.
The sequence identity percentages were higher for T3 than for T7,
suggesting that YeO3-12 is more closely related to T3 than to T7.
This was further supported by the fact that YeO3-12-specific antiserum neutralized T3 infection but not T7 infection. The evidence conclusively indicated that YeO3-12 is the first close relative of
phage T3 to be described. The nucleotide sequence of YeO3-12 (unpublished data) does confirm these conclusions.
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ACKNOWLEDGMENTS |
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Turku Graduate School for Biomedical Sciences, the National Technology Agency, and the Academy of Finland are thanked for financial support.
Jukka Hellman is acknowledged for N-terminal sequencing, and
Hans-Wolfgang Ackermann is acknowledged for the electron microscopy of
the YeO3-12 particles. We thank Erkki Eerola and Kirsti Tuomela for
HPLC analysis of the fatty acids, Elise Ervelä for LPS
degradation studies, Anne Peippo for phage purifications and DNA
isolations, and Jyri Kurkinen for digital art images. Ian Molineux's
help with bacterial strains, phages, and personal communication is greatly appreciated.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Medical Biochemistry and Molecular Biology, Institute of Biomedicine, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland. Phone: 358 2 333 7444. Fax: 358 2 333 7229. E-mail: maria.pajunen{at}utu.fi.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Journal of Bacteriology, September 2000, p. 5114-5120, Vol. 182, No. 18
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