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The LYSOGENIC CYCLE OF THE FILAMENTOUS PHAGE Cflt from Xanthomonas campestris pv. citri
VIROLOGY
156, 305-3 12 (1987)
The Lysogenic Cycle of the Filamentous
Phage Cflt from Xanthomonas campestris pv. citri
TSONG-TEH KUO,*,’ YU-HUEI LIN,* CHUNG-MING HUANG,t SHAU-FENG CHANG,t HWA DAI,* AND TENG-YUNG FENG* *Institute
of Botany,
and tCentral
Laboratory
Received
March
for Molecular 2 1, 1986;
Biology, accepted
Academia October
Sinica,
Taipei,
Republic
of China
13, 1986
A phage, Cflt, forming turbid plaques, was isolated from Xanthomonas campestris pv. cifri. After infection, infected sensitive cells become immune to Cflt and produce very few phages. These properties were genetically rather stable. The phage was purified and shown to be filamentous with a size of 1157 f 73 nm. The genome size is about 7.62 kb. The phage does not affect the growth of host bacteria. Under natural cultivation conditions Cflt-lysogenized cells could be induced spontaneously to give high phage yields, or cured to give phage-free cells. The integration of Cflt DNA into host DNA was proved by Southern blot hybridization. The lysogenic phage was genetically stable in log phase cells and persisted in stationary phase cells through many cell generations in the absence of extracellular phage reinfection. 8 1997 Academic
Press, Inc.
INTRODUCTION
companying paper describes an independently isolated but very closely related phage, Cf16 (Dai et al., 1987).
Xanthomonas campestris pv. citri is a pathogenic bacterium causing citrus canker in orange trees. To study the genetics and the pathogenicity of this bacterium at the molecular level, it is important to have a vector system. Among temperate phages that grow on this organism, one phage that formed a tiny turbid plaque and could lysogenize its host bacterium attracted our attention. The phage was purified and shown to be filamentous. A similar phage, Cf, grows on the same host (Dai et al., 1980). Although the two phages are serologically indistinguishable, Cf forms clear plaques and cannot lysogenize its host. Since the new phage forms turbid plaques and can lysogenize its host, it was named Cflt. Phage life cycles fit into two distinct categories: the lytic and the lysogenic. The filamentous phages are small viruses that contain a circular single-stranded DNA molecule. The DNA is encapsulated in a long protein coat (Denhardt et a/., 1978; Baas, 1985). Unlike other phages, infection with a filamentous phage leads neither to lysis nor to lysogeny of the host cell. Instead, the infected cell extrudes progeny phage particles, while continuing to grow and to produce viable daughter cells (Hsu, 1968; Marvin and Hohn, 1969; Merrian, 1977). Although the life cycle of filamentous phages departs significantly from that of the other phages, they are usually considered to be virulent because a lysogenie cycle has not previously been observed. However, in this investigation we describe a filamentous phage that can engage in a lysogenic cycle. The ac’ TO whom
requests
for reprints
should
MATERIALS
AND
METHODS
Bacterial strains and phages Xanthomonas campestris pv. citriXW47-1 1 was provided by Professor Wen C. Wu, National Chung-Hsing University. Cflt was isolated from X. campestris pv. citri, formed turbid plaques, and could lysogenize its host cells. Cf was isolated from the same host, but formed clear plaques and could not lysogenize its host cells. The LB medium used was described by Bukhari and Metlay (1973). Preparation
of bacterial culture and phages
The bacterial cells were streaked on LB plates and grown at 30” for 48 hr. These plates were then stored at 4”. Phage Cflt and Cf were harvested from semiconfluent lysis on double-layer LB plates. Distilled water was added to the plates, which were kept overnight in a cold room. The phage solution was collected and the cell debris was removed by centrifugation at 10,000 g for 30 min at 4”. The phage titer was assayed by the double-layer agar method (Eisenstark, 1967). The phage was stored at 4’ for use. Assays for immunity, and phage yield
phage infectivity,
Samples taken from the culture were spread onto LB agar plates. After colony formation, a single colony was chosen and transferred to LB broth for 24 hr at 30”. For assay of immunity a sample from this culture
be addressed. 305
0042-6822187
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Copyright Q 1987 by Academic Press. Inc. All rights of reproduction in any form reserved.
KU0 ET AL.
306
was mixed with broth soft agar and poured onto an LB plate. Cflt suspension (0.01 ml) was spotted onto the layer containing bacteria to be tested. Sensitive cells formed a clear zone and immune cells did not. For determination of phage infectivity and phage yield the sample from the above culture was centrifuged at 10,000 g for 20 min to remove cell debris. The O.Olml samples from dilutions of the supernatant were plated with sensitive bacteria. Determination of the growth curve for bacteria and the K value of Cflt antiserum Bacteria were suspended in LB at a concentration of 1 X 1 O* cells/ml and incubated at 30°, with shaking. At different intervals, samples were diluted and spread on LB agar plates. After colony formation the number of colonies was counted. Antiserum against Cflt was prepared by the procedure described by Williams and Chase (1967). The K value was determined by the method described by Adams (1959). Purification
of phages
For the purification of Cflt an overnight infected culture was harvested, and host cells and debris were removed by centrifugation at 6000 g for 10 min. Solid NaCl was added to the supernatant to a final concentration of 0.05 M, and polyethyene glycol 6000 was then added to a final concentration of 3%. After thorough stirring, the mixture was allowed to settle overnight in a cold room. The precipitate was collected by centrifugation at 6000 g for 10 min and then resuspended in HiO. Further purification was carried out by centrifugation through a CsCl step density gradient, (1.18, 1.25, 1.29, 1.34, and 1.39 g/ml) in a Beckman SW41 swinging bucket rotor at 23,000 rpm for 22 hr at 5”. Electron microscopy The phage suspension was mixed with an equal volume of 2% phosphotungstic acid. A drop of this mixture was applied to a grid coated with Formvar and carbon. After the excess solution was removed with filter paper, the specimen was examined in an electron microscope. Purification
of phage DNA
A purified phage suspension was dialyzed against TEN (25 mMTris-HCI; 10 mM EDTA and 0.15 M NaCI, pH 8.5). The phage protein coat was then dissociated by adding sodium dodecyl sulfate (SDS) to a final concentration of 2%. The proteins were further digested with Pronase (1 mg/ml) at 60” for 18 hr or overnight. Contaminating RNA was digested by the addition of
RNase (50 pg/ml) and incubated at 37” for 1 hr. NaCIO, was added to a final concentration of 1 M and an equal volume of PIC (TEN-saturated mixture of phenol:isoamyl alcohol:chloroform (50:2:48) was then added. This mixture was shaken thoroughly for 15 min. After 5 min centrifugation at 9000 g, the aqueous phase was collected and the phage DNA was precipitated with ethyl alcohol. The pelleted phage DNA was then dissolved in TEN and dialyzed against two changes of TEN with 2 M NaCI, and then finally against TEN. Preparation of the replicative Cf and Cflt infected cells
form (RF) DNA from
The bacterial cells were grown in 500 ml PS medium. The cells, at a density of 2 X 10’ cells/ml, were infected with Cf or Cflt at a multiplicity of 20 and treated with 170 pg/ml chloramphenicol at 10 min postinfection. Chloramphenicol was added to increase the synthesis of RF DNA and to prevent chromosomal DNA synthesis (Clewell, 1972). After a 4-hr incubation at 28”, the infected cells were harvested, chilled, washed once with 250 ml buffer (10 mMTris-HCI, pH 8.0,O.l mM EDTA), and lysed with SDS and NaOH (Birnboim and Doly, 1979). lsopropanol was added to the supernatant. After centrifugation at 27,200 g for 20 min, the pellet was resuspended in 5 ml Tris-glucose (25 mM Tris-HCI, pH 8.0, 10 mM EDTA, 50 mM glucose) and 15 ml of 5 M potassium acetate. The bacterial DNA and debris were spun at 27,200 g for 20 min. The supernatant, containing the RF DNA, was precipitated with 2 vol of ethanol at -20” for 2 to 4 hr and recovered by centrifugation at 12,000 g for 30 min. The pellet was dissolved in 8 ml of TE buffer (10 mM Tris-HCI, pH 8.0, 1 mM EDTA). The DNA was then purified by centrifugation to equilibrium in a CsCl ethidium bromide density gradient. The DNA thus prepared was used directly for restriction endonuclease digestion. Isolation of lysogen DNA Logarithmically growing lysogenic cells at 2 to 4 10’ cells/ml were harvested by centrifugation, washed with 5 ml of 100 mM Tris-HCI, 10 mM NaCl at pH 7.6, and suspended in 1.8 ml of 50 mM TrisHCI (pH 7.6) 100 mM NaCI, and 5 mM disodium EDTA. The washed cell suspension was lysed by the addition of lysozyme solution (10 mg/ml in HZ0 10 min at room temperature) and 0.2 ml of 20% SDS to the buffered cell suspension. The lysed cell suspension was extracted with phenol:chloroform:isoamyl alcohol (24:24: 1 v/v) until no interface could be detected between the aqueous and organic phases. The aqueous phase was precipitated with 2 vol of cold (-20”) ethanol. The ethanol precipitate was collected by centrifugation at X
LYSOGENIC CYCLE OF X. csmpestris
13,000 g for 10 min, dried in vacua, and suspended in 2 ml of 50 mM Tris-HCI (pH 7.6) 100 mM NaCI, and 5 rnM disodium EDTA. This solution was incubated with 200 U RNase A at room temperature for 4 hr. The RNase-treated solution was extracted three times with phenol:chloroform:isoamyl alcohol. The aqueous phase was precipitated twice with ethanol, and the dried precipitate was suspended in 10 mM Tris-HCI (pH 7.6) and 0.1 mM disodium EDTA. Restriction
endonucleases
Restriction endonucleases were purchased from New England BioLabs and the manufacturer’s detailed procedures were used as described. Before loading into sample wells, the restriction digests (20 ~1) were heated to 70” for 10 to 15 min. Agarose gel electrophoresis Samples of restriction enzyme-treated DNA were loaded into wells in horizontal 0.6 to 1.5% agarose slab gels prepared in electrophoresis buffer and with 0.25 pg of ethidium bromide/ml. Electrophoresis was carried out at 25 to 100 V for 6 to 20 hr. After electrophoresis, the restriction profiles were photographed during illumination with UV light with a transilluminator. Transfer of DNA to Gene-screen hybridization transfer membrane and hybridization Native DNA in agarose gels was denatured in situ and transferred by blotting onto a Gene-screen hybridization transfer membrane as described by Southern (1975). The prepared membrane was placed in polyethylene bags to which a prehybridization mixture consisting of 0.1% polyvinylpyrrolidone (mol wt 40,000), 0.1% bovine serum albumin, 0.1% Ficoll (mol wt 400,000), and denatured calf thymus DNA (100 pg/mI) was added. The bags were sealed and incubated with constant agitation at 60”, overnight. The prehybridization mixture was replaced with a hybridization mixture that contained 0.3 M NaCI, 0.06 M Tris-HCI (pH 8.0) 0.002 M EDTA, 0.02% polyvinylpyrrolidone (mol wt 40,000), 0.02% bovine serum albumin, 0.02% Ficoll (mol wt 400,000), 1% SDS, denatured calf thymus DNA (100 pg/ml), and 5 X 1 O6to 5 X 1O7 dpm of denatured (100” for 10 min) radioactive probe in a total volume of 50 PI/cm2 of hybridization transfer membrane. The bags were resealed and incubated, with constant agitation, at 60” for 18 to 24 hr. After hybridization the membrane was washed twice in 0.3 M NaCI, 0.06 M Tris-HCI (pH 8.0) 0.002 M EDTA at room temperature; twice in 0.06 M Tris-HCI (pH 8.0) 0.02 M EDTA, and 1% SDS at 60” for 30 min with constant agitation; then twice in 0.03 M NaCI, 0.006 M Tris-HCI (pH 8.0)
PHAGE
307
0.0002 M EDTA at room temperature for 30 min with constant agitation, air-dried, and analyzed by autoradiography. RESULTS Characterization
of Cflt
The phage was first isolated through three stages of single plaque purification, then further purified through polyethylene glycol 6000 precipitation and CsCl step gradient as described under Materials and Methods. A sharp visible band located in the middle of the gradient and corresponding to the phage activity was obtained. By examination under electron microscopy, the purified phage was identified as a filamentous phage with a length of 1157 + 73 nm and a diameter of 6 nm (Fig. 1). Since Cflt was morphologically similar to the other filamentous phage, Cf, isolated from the same host bacteria (Dai et a/., 1980) some important properties were compared. The K value of Cflt antiserum against the two phages is identical, 1 X 10 min. When native DNAs from both phages were purified and analyzed by agarose gel electrophoresis, the DNA bands migrated in the same pattern and position (data not shown). There is no detectable difference in the size and form of Cflt DNA and Cf DNA, which is single stranded and circular (Dai et al., 1980). The RF DNAs from both phages were isolated and digested with BarnHI and Hincll, respectively. BarnHI cut Cflt RF into one fragment and Hincll cut it into four fragments. The digestion patterns for Cflt and Cf were identical (Fig. 2). From the size markers the molecular size of Cflt was determined to be about 7.62 kb (Yang and Kuo, 1984). Based on the above data the two phages are nearly identical. However, there are some differences between these phages: Cf forms clear plaques (Fig. 3) and does not lysogenize its host, whereas the Cflt forms turbid plaques (Fig. 3) and can lysogenize its host. Immunity and phage production infected cells
in Cflt-
Host bacteria at a concentration of 8 X 10’ cells/ml were infected with Cflt at a multiplicity of 100. After 90 min. incubation, the cells were spread on an LB agar plate. After colony formation, 400 were chosen randomly; 90% of these were determined to be infected. Cells from 200 of the infected colonies were tested for immunity to Cflt or to Cf, with the results shown in Table 1. Seventy percent were immune to Cflt while 30% were sensitive. Nearly all were sensitive to Cf. A lawn of Cflt-infected cells did not form plaques with Cflt but it did with Cf. Thus, Cflt infection produces immunity
308
KU0
FIG. 1. Electron
micrograph
of phage
Cflt.
ET
The phages
to Cflt, but not to Cf, in roughly 70% of the infected cells. Immunity of Cflt-infected cells to superinfection with Cflt is correlated with another property, the yield of phage from the infected cells, shown in Table 1. Cfltinfected cells can be divided into high yielders, pro123456
FIG. 2. Determination of the molecular size of Cflt DNA. Cflt RF DNA was digested with BarnHI and Hincll, respectively. Digested DNA fragments were electrophoresed horizontally for 12 hr in a 1% agarose gel at 40 V in TBE buffer. X DNA digested with HindIll was used as size marker. Cf RF DNA digested with same enzyme was used for comparison. Lane 1, X DNA digested with HindIll and EcoRI; lane 2, Cflt RF DNA digested with BarnHI; lane 3, Cf RF DNA digested with BarnHI; lane 4. Cflt RF DNA digested with Hincll; lane 5, Cf RF DNA digested with Hincll; lane 6. X DNA digested with HindIll.
AL.
were
stained
with
2% phosphotungstic
acid
ducing a phage titer over 10’ PFU/ml, and low yielders, producing a phage titer below 1O7 PFU/ml. Cflt-immune cells were predominantly low yielders (959/o) while sensitive cells were predominantly high yielders (91%). Stability of immunity
and phage yield
These properties were tested by transferring 30 colonies of each type through five passages, isolating single colonies at each passage. Among immune colonies, the original properties of immunity and low phage yield were completely retained. Among sensitive col-
FIG. 3. Plaque
morphology
of Cf (left) and Cflt (right)
LYSOGENIC
CYCLE
OF
X. campesrris
TABLE
PHAGE
309
1
IMMUNITY AND PHAGE YIELD IN Cflt-INFECTED
CELLS
Immunity
Immune
Sensitive
to Cflt
to Cflt
Phage
No. of cells obtained
%
141
70
Low phage High phage
59
30
Low phage High phage
yield No. of cells obtained
%
yielder yielder
131 8
95 5
yielder yielder
6 53
9 91
onies, 28 retained their original properties while 2 became free of phage.
original properties, phage yielders.
Effect of lysogeny on growth
Induction
The lysogenic cells were grown in LB medium and at different times bacterial growth and phage yield were measured (Fig. 4). The lysogen grew at the same rate as uninfected bacteria and the phage yield did not increase.
Phage production in lysogenized cell cultures was always between 1O6 and 10’ PFU/ml. However, when these cultures were grown and transferred several times, the phage titer increased to 10g-lO’o PFU/ml. We suspected this was due to the conversion of low yielders into high yielders. To test this possibility, a single lysogen was grown in LB and samples were taken at various times to determine the proportion of low phage yielders, high phage yielders, and cured cells. The results are summarized in Table 2. Three independent experiments gave vet-y similar results. Over a 48-hr period, 21% of the low phage yielders were converted to high phage yielders and 8% were cured. The cured cells were sensitive to Cflt infection.
The persistence
of prophage
in Cflt-infected
cells
The ability of immune cells to produce phage, after growth in LB containing antiserum, was determined. Figure 5 shows that the antiserum effectively killed all phages released into the medium, preventing reinfection. After growth for 24 generations in antiserum-containing medium, 100 individual colonies were tested for immunity and phage yield. Ninety-five retained their
I
and curing of Cflt lysogens
I
1
I
I
I
I
I
I
2
4
6
6
10
12
14
16
TIME
3 were cured, and 2 were high
1
I 20
(h)
FIG. 4. Growth of Cflt-lysogenized cells and phage production in LB medium at 30” with shaking. phage titer were measured as described under Materials and Methods. 0, Growth of Cflt lysogenized A, Phage production in a lysogen culture.
The number of bacterial cells and the cells. 0, Growth of uninfected bacteria.
310
KU0 IO’
= 5 g
ET AL.
tion of Cflt DNA into host DNA occurs, the DNAs from Cflt-infected cells, Cf-infected cells, noninfected host cells, and Cflt RF were isolated and digested with BarnHI, HindIll, and Pvull, respectively. The resulting fragments were fractionated by agarose gel electrophoresis. The DNA fragments in the gel were then denatured in situ, transferred to a hybridization membrane, and hybridized to Cflt RF DNA that had been labeled with [32P]lTP by nick translation (Rigby et a/., 1977). The location of viral sequences among the fragments of bacterial chromosome DNA was determined by autoradiography. The results in Fig. 6 show that Cflt DNA hybridized only with chromosomal DNA isolated from Cflt-infected cells, not with DNA from uninfected host cells. Since the only band detected was in the position of host chromosome DNA it appears that there was no free form of phage DNA in the Cflt-infected cell (Fig. 6, lanes 1, 2). Two bands from Cflt RF DNA were detected; one is supercoiled (RF I) and another, open circular (RF II). When Cflt RF was isolated from newly infected cells, both forms of RF were usually found. When RF DNA was cut with HindIll, two fragments of 5.7 and 1.6 kb were produced. When the DNA from Cflt-infected cells was digested with the same enzymes, three fragments of 4.6, 2.9, and 1.6 kb were obtained. The 5.7-kb fragment was replaced by two new fragments (Fig. 6, lanes 5, 10). To confirm this observation two other restriction enzymes, Pvull and BarnHI, were tested with similar results. m/ull cut Cflt RF into two fragments of 5.2 and 2.4 kb, but cleaved Cflt-infected cell DNA into three fragments of 5.2, 2.3, and 1.3 kb. The 5.2-kb fragment was preserved and the 2.4-kb fragment was replaced by two new fragments of 2.3 and 1.3 kb (Fig. 6, lanes 6, 11). BarnHI has one site on RF DNA. When the DNA isolated from Cflt-infected cells was cleaved by the same enzyme, the Cflt RF DNA fragment was missing, and two bands of 15.6 and 14.4 kb were detected. On this gel the two bands are so close that only one thicker band was observed (Fig. 6, lanes 4, 9). These data provide the
r
lo5
10'
a tw F w
io3
F! lo*
10'
loo 5
13
19
24
29
36
42
46
TIME(h) FIG. 5. Persistance of prophage in the presence of Cflt antiserum. Lysogen was grown in 8 ml LB broth to which previously 4 ml of 10-l antiserum against Cflt had been added. For control the antiserum was omitted. The cultures were incubated at 30”. At different time intervals, 1 ml of cultured cells was taken to be centrifuged at 10,000 g for 20 min. The phage titer in supernatant was determined. 0, Phage titer in lysogenic culture with antiserum. 0, Phage titer in lysogenic culture without antiserum.
These figures, 21% high yielders and 8% cured cells, are in good agreement with the proportion of these classes formed following Cflt infection (Table 1). Physical evidence of the integration into host chromosome DNA
of Cflt DNA
The stability of phage yield and immunity to superinfection suggests that each daughter cell inherited the phage genome faithfully during cell division. Cells may have transmitted the phage DNA either as a plasmid (Ikeda and Tomizawa, 1965) or as an integrated prophage (Hershey, 1971). To determine whether integraTABLE
2
INDUCTION AND CURING OF Cflt LYSOGEN UNDER NATURAL CULTIVATION Incubation Types
of cells
(%I
0
Phage free cell High phage yielder Low phage yielder
Note. A pure lysogen samples medium.
from this culture The phage yield
8 0 0
100 was
0 0 100
time
CONDITIONS
(hr)
16
24
32
0 0 100
1
3
3 96
10
87
40
48
4 12 84
8 21 71
selected by single colony isolation, transferred to LB medium, and grown at 30” with shaking. At different times were spread on an LB agar plate. After colony formation, 200 colonies were tested by transfer into 1 ml of LB in each culture was determined as described under Materials and Methods.
LYSOGENIC
1 2 3 4 5 6 7 6 9 10 11
CYCLE
12
FIG. 6. Integration of Cflt DNA into host chromosome. The DNAs from Cflt RF, Cflt lysogen, Cf infected cells, and noninfected host cells were digested with BarnHI, Hindlll, and Pvull. All digests were electrophoresed horizontally for 16 hr in a 0.8% agarose gel at 30 V in TEE buffer, blotted, and hybridized with nick-translated 32P-labeled Cflt RF DNA. X DNA digested with HindIll was used as size markers. The amount of DNA used in each sample was 0.5 rg for X DNA, 0.2 pg for Cflt RF DNA and 20 pg for lysogen DNA, Cf infected cells and host cells. Lane 1, DNA from uninfected host cells; lane 2, undigested DNA from Cflt lysogen; lane 3, DNA from uninfected host cells digested with BarnHI; lane 4, DNA from Cflt lysogen digested with BarnHI; lane 5, DNA from Cflt lysogen digested with HindIll; lane 6, DNA from Cflt lysogen digested with Pvull; lane 7, DNA from Cf infected cells; lane 8, Cflt RF DNA; lane 9, Cflt RF DNA digested with BarnHI; lane 10, Cflt RF DNA digested with HindIll; lane 11, Cflt RF DNA digested with h/VII; lane 12, X DNA digested with HindIll.
OF X.
campestris
PHAGE
311
growth of infected cells, and has a low phage yield, whereas Cf forms clear plaques, slows the growth of infected cells, and has a high yield. Moreover, Cflt infection results in the formation of lysogens immune to superinfection by Cflt, while infection by Cf does not produce lysogens. To date, lysogens are known to be produced only by viruses containing double-stranded DNA. This is the first report of lysogenization by a virus containing circular single-stranded DNA. Cflt-infected bacteria can be divided into two types on the basis of immunity and phage yield. In a typical infection, about 70% of the infected cells become immune, low yielders while 30% are sensitive, high yielders. Single colony isolation of infected cells led to the conclusion that each type was relatively stable, except that after repeated transfer, the lysogenic type of cell could be converted to the sensitive high yielder (20%) or cured (8%). The very low production of phage by lysogens was confirmed by our inability to find Cflt RF DNA in lysogenized cells. We do not believe that the conversion of immune, low yielders to sensitive, high yielders is an artifact due to contamination of the cultures. If this were true, we should have detected the contaminating cells at an early stage, since both lysogens and cured cells grow at the same rate (Table 2). We have tried to induce or to cure lysogens with UV light, heat, ethidium bromide, and acridine orange, without success. Similar failures have been observed 12345676
evidence that the Cflt DNA was integrated into the host DNA. To study the frequency and the pattern of Cflt DNA integration, 15 infected colonies were randomly picked, and the DNAs from these isolates were digested with BarnHI and hybridized with 32P-labeled Cflt RF DNA. The result is shown in Fig. 7. All Cflt lysogens contained integrated phage DNA. Two bands were detected in all Cflt lysogens. Since the position of the two samesized bands was always identical, it means that one copy of Cflt DNA is integrated at same site on host DNA. DISCUSSION
Comparison of virion morphology, serology, and the size and form of native viral DNA showed phage Cflt to be similar to Cf, a previously characterized phage containing single-stranded circular DNA (Dai et a/., 1980). However, the two phages could be distinguished: Cflt forms turbid plaques, does not affect the
FIG. 7. The integration pattern of viral DNA in Cflt lysogens. DNAs were isolated from Cflt lysogens and digested with BarnHI. The conditions for gel electrophoresis and Southern hybridization were described as in the legend of Fig. 6. Lane 1, Cflt RF DNA; lane 2, Cflt RF DNA digested with BarnHI; lane 3, DNA from Cflt lysogen P41, digested with BarnHI; lane 4, DNA from Cflt lysogen P37, digested with BarnHI; lane 5, DNA from Cflt lysogen P31, digested with BarnHI; lane 6, ONA from Cflt lysogen d4, digested with BarnHI; lane 7, DNA from Cflt lysogen 1 1, digested with BarnHI; lane 8, X DNA digested with HindIll.
312
KU0
in other lysogenic systems (Barksdale and Arden, 1974; Orndorff et al., 1983). Growth of Cflt lysogens through viral antiserum proves that both immunity and ability to produce phage are stable properties of the lysogens, not due to reinfection. Two kinds of stable lysogens of bacterial viruses are known. In one, the phage DNA is integrated into the bacterial chromosome (Hershey, 1971). In the other, the phage DNA is a plasmid whose replication is coordinated with that of the chromosome (Ikeda and Tomizawa, 1965). Our Southern blots demonstrate that Cflt DNA is integrated into the host chromosome. The band patterns produced by cutting individual lysogens with different restriction enzymes (Figs. 6 and 7) allow us to conclude that each lysogenic cell has only one integration site and that the site is at the same chromosomal location in each lysogen. ACKNOWLEDGMENTS We thank Wen C. Wu for providing us X. campestris pv. citri strain XW47-11. The work was supported by a grant from National Science Council, Republic of China.
REFERENCES ADAMS, M. H. (1959). Antigenic properties. In “Bacteriophages,” pp. 97-l 17. Wiley Interscience, New York. BAAS, P. D. (1985). DNA replication of single-stranded Escherichia co/i DNA phages. Biochim. Biophys. Acta 825, 1 1 l-l 39. BARKSDALE, L., and ARDEN, S. B. (1974). Persisting bacteriophage infections, lysogeny, and phage conversions. Annu. Rev. Microbial. 28,265-229. BIRNBOIM, H. C., and DOLY, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513-l 523.
ET AL. BUKHARI, A. I., and METLAY, M. (1973). Genetic mapping of prophage Mu. Virology 54, 109-l 16. CLEWELL, D. B. (1972). Nature of Col El plasmid replication in the presence of chloramphenicol. J. Bacterial, 110, 667-676. DAI, H., CHIANG, K. S., and Kuo, T. T. (1980). Characterization of a new filamentous phage Cf from Xanthomonas citri. /. Gen. Viral. 46,277-289. DAI, H., TSAY, S.-H., Kuo, T.-T., LIN, Y.-H., and Wu, W. C. (1987). Neolysogenization of Xanthomonas campestris pv. citri infected with filamentous phage Cfl6. Virology 156, 313-320. DENHARDT, D. T., Rnv, D. S., and DRESSLER, D. (1978). “Single-Stranded DNA Phages.” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. EISENSTARK, A. (1967). Bacteriophage techniques. /n “Methods in Virology” (K. Maramorosch and H. Koprowaki, Eds.), Vol. 1, pp. 449-524. Academic Press, New York. HERSHEY, A. D. (1971). “The Bacteriophage Lambda,” p, 792. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. HSU, Y. C. (1968). Propagation or elimination of viral infection in carrier cells. Bacteria/ Rev. 32, 387-391, IKEDA, H., and TOMIZAWA. J. (1965). Transducing fragments in generalized transduction by phage P1.J. Mol. Biol. 14, 85-109. MARVIN, D. A., and HOHN, B. (1969). Filamentous bacterial viruses. Bacterial. Rev. 33, 172-209. MERRIAN, V. (1977). Stability of the carrier state in bacteriophage M 13. infected cells. J. Viral. 21, 880-888. ORNDORFF, P., STELLWAG. E., STARICH, T.. DWORKIN, M., and ZISSLER, J. (1983). Genetic and physical characterization of lysogeny by bacteriophage MX8 in Myxococcus xanthus. J. Bacterial. 154, 772779. RIGBY, P. W. J., DIECKMAN, M., RHODES, C., and BERG, P. (1977). Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113, 237251. SOUTHERN, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. f. Mol. Biol. 98, 501517. WILLIAMS, C. A., and CHASE, M. W. (Eds.) (1967). “Methods in Immunology and Immunochemistry,” Vol. 1, pp. 15-l 20. Academic Press, New York. YANG, M. K., and Kuo. T. T. (1984). A physical map of the filamentous bacteriophage Cf genome. 1. Gen. Viral. 65, 1175-l 181.
156, 305-3 12 (1987)
The Lysogenic Cycle of the Filamentous
Phage Cflt from Xanthomonas campestris pv. citri
TSONG-TEH KUO,*,’ YU-HUEI LIN,* CHUNG-MING HUANG,t SHAU-FENG CHANG,t HWA DAI,* AND TENG-YUNG FENG* *Institute
of Botany,
and tCentral
Laboratory
Received
March
for Molecular 2 1, 1986;
Biology, accepted
Academia October
Sinica,
Taipei,
Republic
of China
13, 1986
A phage, Cflt, forming turbid plaques, was isolated from Xanthomonas campestris pv. cifri. After infection, infected sensitive cells become immune to Cflt and produce very few phages. These properties were genetically rather stable. The phage was purified and shown to be filamentous with a size of 1157 f 73 nm. The genome size is about 7.62 kb. The phage does not affect the growth of host bacteria. Under natural cultivation conditions Cflt-lysogenized cells could be induced spontaneously to give high phage yields, or cured to give phage-free cells. The integration of Cflt DNA into host DNA was proved by Southern blot hybridization. The lysogenic phage was genetically stable in log phase cells and persisted in stationary phase cells through many cell generations in the absence of extracellular phage reinfection. 8 1997 Academic
Press, Inc.
INTRODUCTION
companying paper describes an independently isolated but very closely related phage, Cf16 (Dai et al., 1987).
Xanthomonas campestris pv. citri is a pathogenic bacterium causing citrus canker in orange trees. To study the genetics and the pathogenicity of this bacterium at the molecular level, it is important to have a vector system. Among temperate phages that grow on this organism, one phage that formed a tiny turbid plaque and could lysogenize its host bacterium attracted our attention. The phage was purified and shown to be filamentous. A similar phage, Cf, grows on the same host (Dai et al., 1980). Although the two phages are serologically indistinguishable, Cf forms clear plaques and cannot lysogenize its host. Since the new phage forms turbid plaques and can lysogenize its host, it was named Cflt. Phage life cycles fit into two distinct categories: the lytic and the lysogenic. The filamentous phages are small viruses that contain a circular single-stranded DNA molecule. The DNA is encapsulated in a long protein coat (Denhardt et a/., 1978; Baas, 1985). Unlike other phages, infection with a filamentous phage leads neither to lysis nor to lysogeny of the host cell. Instead, the infected cell extrudes progeny phage particles, while continuing to grow and to produce viable daughter cells (Hsu, 1968; Marvin and Hohn, 1969; Merrian, 1977). Although the life cycle of filamentous phages departs significantly from that of the other phages, they are usually considered to be virulent because a lysogenie cycle has not previously been observed. However, in this investigation we describe a filamentous phage that can engage in a lysogenic cycle. The ac’ TO whom
requests
for reprints
should
MATERIALS
AND
METHODS
Bacterial strains and phages Xanthomonas campestris pv. citriXW47-1 1 was provided by Professor Wen C. Wu, National Chung-Hsing University. Cflt was isolated from X. campestris pv. citri, formed turbid plaques, and could lysogenize its host cells. Cf was isolated from the same host, but formed clear plaques and could not lysogenize its host cells. The LB medium used was described by Bukhari and Metlay (1973). Preparation
of bacterial culture and phages
The bacterial cells were streaked on LB plates and grown at 30” for 48 hr. These plates were then stored at 4”. Phage Cflt and Cf were harvested from semiconfluent lysis on double-layer LB plates. Distilled water was added to the plates, which were kept overnight in a cold room. The phage solution was collected and the cell debris was removed by centrifugation at 10,000 g for 30 min at 4”. The phage titer was assayed by the double-layer agar method (Eisenstark, 1967). The phage was stored at 4’ for use. Assays for immunity, and phage yield
phage infectivity,
Samples taken from the culture were spread onto LB agar plates. After colony formation, a single colony was chosen and transferred to LB broth for 24 hr at 30”. For assay of immunity a sample from this culture
be addressed. 305
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$3.00
Copyright Q 1987 by Academic Press. Inc. All rights of reproduction in any form reserved.
KU0 ET AL.
306
was mixed with broth soft agar and poured onto an LB plate. Cflt suspension (0.01 ml) was spotted onto the layer containing bacteria to be tested. Sensitive cells formed a clear zone and immune cells did not. For determination of phage infectivity and phage yield the sample from the above culture was centrifuged at 10,000 g for 20 min to remove cell debris. The O.Olml samples from dilutions of the supernatant were plated with sensitive bacteria. Determination of the growth curve for bacteria and the K value of Cflt antiserum Bacteria were suspended in LB at a concentration of 1 X 1 O* cells/ml and incubated at 30°, with shaking. At different intervals, samples were diluted and spread on LB agar plates. After colony formation the number of colonies was counted. Antiserum against Cflt was prepared by the procedure described by Williams and Chase (1967). The K value was determined by the method described by Adams (1959). Purification
of phages
For the purification of Cflt an overnight infected culture was harvested, and host cells and debris were removed by centrifugation at 6000 g for 10 min. Solid NaCl was added to the supernatant to a final concentration of 0.05 M, and polyethyene glycol 6000 was then added to a final concentration of 3%. After thorough stirring, the mixture was allowed to settle overnight in a cold room. The precipitate was collected by centrifugation at 6000 g for 10 min and then resuspended in HiO. Further purification was carried out by centrifugation through a CsCl step density gradient, (1.18, 1.25, 1.29, 1.34, and 1.39 g/ml) in a Beckman SW41 swinging bucket rotor at 23,000 rpm for 22 hr at 5”. Electron microscopy The phage suspension was mixed with an equal volume of 2% phosphotungstic acid. A drop of this mixture was applied to a grid coated with Formvar and carbon. After the excess solution was removed with filter paper, the specimen was examined in an electron microscope. Purification
of phage DNA
A purified phage suspension was dialyzed against TEN (25 mMTris-HCI; 10 mM EDTA and 0.15 M NaCI, pH 8.5). The phage protein coat was then dissociated by adding sodium dodecyl sulfate (SDS) to a final concentration of 2%. The proteins were further digested with Pronase (1 mg/ml) at 60” for 18 hr or overnight. Contaminating RNA was digested by the addition of
RNase (50 pg/ml) and incubated at 37” for 1 hr. NaCIO, was added to a final concentration of 1 M and an equal volume of PIC (TEN-saturated mixture of phenol:isoamyl alcohol:chloroform (50:2:48) was then added. This mixture was shaken thoroughly for 15 min. After 5 min centrifugation at 9000 g, the aqueous phase was collected and the phage DNA was precipitated with ethyl alcohol. The pelleted phage DNA was then dissolved in TEN and dialyzed against two changes of TEN with 2 M NaCI, and then finally against TEN. Preparation of the replicative Cf and Cflt infected cells
form (RF) DNA from
The bacterial cells were grown in 500 ml PS medium. The cells, at a density of 2 X 10’ cells/ml, were infected with Cf or Cflt at a multiplicity of 20 and treated with 170 pg/ml chloramphenicol at 10 min postinfection. Chloramphenicol was added to increase the synthesis of RF DNA and to prevent chromosomal DNA synthesis (Clewell, 1972). After a 4-hr incubation at 28”, the infected cells were harvested, chilled, washed once with 250 ml buffer (10 mMTris-HCI, pH 8.0,O.l mM EDTA), and lysed with SDS and NaOH (Birnboim and Doly, 1979). lsopropanol was added to the supernatant. After centrifugation at 27,200 g for 20 min, the pellet was resuspended in 5 ml Tris-glucose (25 mM Tris-HCI, pH 8.0, 10 mM EDTA, 50 mM glucose) and 15 ml of 5 M potassium acetate. The bacterial DNA and debris were spun at 27,200 g for 20 min. The supernatant, containing the RF DNA, was precipitated with 2 vol of ethanol at -20” for 2 to 4 hr and recovered by centrifugation at 12,000 g for 30 min. The pellet was dissolved in 8 ml of TE buffer (10 mM Tris-HCI, pH 8.0, 1 mM EDTA). The DNA was then purified by centrifugation to equilibrium in a CsCl ethidium bromide density gradient. The DNA thus prepared was used directly for restriction endonuclease digestion. Isolation of lysogen DNA Logarithmically growing lysogenic cells at 2 to 4 10’ cells/ml were harvested by centrifugation, washed with 5 ml of 100 mM Tris-HCI, 10 mM NaCl at pH 7.6, and suspended in 1.8 ml of 50 mM TrisHCI (pH 7.6) 100 mM NaCI, and 5 mM disodium EDTA. The washed cell suspension was lysed by the addition of lysozyme solution (10 mg/ml in HZ0 10 min at room temperature) and 0.2 ml of 20% SDS to the buffered cell suspension. The lysed cell suspension was extracted with phenol:chloroform:isoamyl alcohol (24:24: 1 v/v) until no interface could be detected between the aqueous and organic phases. The aqueous phase was precipitated with 2 vol of cold (-20”) ethanol. The ethanol precipitate was collected by centrifugation at X
LYSOGENIC CYCLE OF X. csmpestris
13,000 g for 10 min, dried in vacua, and suspended in 2 ml of 50 mM Tris-HCI (pH 7.6) 100 mM NaCI, and 5 rnM disodium EDTA. This solution was incubated with 200 U RNase A at room temperature for 4 hr. The RNase-treated solution was extracted three times with phenol:chloroform:isoamyl alcohol. The aqueous phase was precipitated twice with ethanol, and the dried precipitate was suspended in 10 mM Tris-HCI (pH 7.6) and 0.1 mM disodium EDTA. Restriction
endonucleases
Restriction endonucleases were purchased from New England BioLabs and the manufacturer’s detailed procedures were used as described. Before loading into sample wells, the restriction digests (20 ~1) were heated to 70” for 10 to 15 min. Agarose gel electrophoresis Samples of restriction enzyme-treated DNA were loaded into wells in horizontal 0.6 to 1.5% agarose slab gels prepared in electrophoresis buffer and with 0.25 pg of ethidium bromide/ml. Electrophoresis was carried out at 25 to 100 V for 6 to 20 hr. After electrophoresis, the restriction profiles were photographed during illumination with UV light with a transilluminator. Transfer of DNA to Gene-screen hybridization transfer membrane and hybridization Native DNA in agarose gels was denatured in situ and transferred by blotting onto a Gene-screen hybridization transfer membrane as described by Southern (1975). The prepared membrane was placed in polyethylene bags to which a prehybridization mixture consisting of 0.1% polyvinylpyrrolidone (mol wt 40,000), 0.1% bovine serum albumin, 0.1% Ficoll (mol wt 400,000), and denatured calf thymus DNA (100 pg/mI) was added. The bags were sealed and incubated with constant agitation at 60”, overnight. The prehybridization mixture was replaced with a hybridization mixture that contained 0.3 M NaCI, 0.06 M Tris-HCI (pH 8.0) 0.002 M EDTA, 0.02% polyvinylpyrrolidone (mol wt 40,000), 0.02% bovine serum albumin, 0.02% Ficoll (mol wt 400,000), 1% SDS, denatured calf thymus DNA (100 pg/ml), and 5 X 1 O6to 5 X 1O7 dpm of denatured (100” for 10 min) radioactive probe in a total volume of 50 PI/cm2 of hybridization transfer membrane. The bags were resealed and incubated, with constant agitation, at 60” for 18 to 24 hr. After hybridization the membrane was washed twice in 0.3 M NaCI, 0.06 M Tris-HCI (pH 8.0) 0.002 M EDTA at room temperature; twice in 0.06 M Tris-HCI (pH 8.0) 0.02 M EDTA, and 1% SDS at 60” for 30 min with constant agitation; then twice in 0.03 M NaCI, 0.006 M Tris-HCI (pH 8.0)
PHAGE
307
0.0002 M EDTA at room temperature for 30 min with constant agitation, air-dried, and analyzed by autoradiography. RESULTS Characterization
of Cflt
The phage was first isolated through three stages of single plaque purification, then further purified through polyethylene glycol 6000 precipitation and CsCl step gradient as described under Materials and Methods. A sharp visible band located in the middle of the gradient and corresponding to the phage activity was obtained. By examination under electron microscopy, the purified phage was identified as a filamentous phage with a length of 1157 + 73 nm and a diameter of 6 nm (Fig. 1). Since Cflt was morphologically similar to the other filamentous phage, Cf, isolated from the same host bacteria (Dai et a/., 1980) some important properties were compared. The K value of Cflt antiserum against the two phages is identical, 1 X 10 min. When native DNAs from both phages were purified and analyzed by agarose gel electrophoresis, the DNA bands migrated in the same pattern and position (data not shown). There is no detectable difference in the size and form of Cflt DNA and Cf DNA, which is single stranded and circular (Dai et al., 1980). The RF DNAs from both phages were isolated and digested with BarnHI and Hincll, respectively. BarnHI cut Cflt RF into one fragment and Hincll cut it into four fragments. The digestion patterns for Cflt and Cf were identical (Fig. 2). From the size markers the molecular size of Cflt was determined to be about 7.62 kb (Yang and Kuo, 1984). Based on the above data the two phages are nearly identical. However, there are some differences between these phages: Cf forms clear plaques (Fig. 3) and does not lysogenize its host, whereas the Cflt forms turbid plaques (Fig. 3) and can lysogenize its host. Immunity and phage production infected cells
in Cflt-
Host bacteria at a concentration of 8 X 10’ cells/ml were infected with Cflt at a multiplicity of 100. After 90 min. incubation, the cells were spread on an LB agar plate. After colony formation, 400 were chosen randomly; 90% of these were determined to be infected. Cells from 200 of the infected colonies were tested for immunity to Cflt or to Cf, with the results shown in Table 1. Seventy percent were immune to Cflt while 30% were sensitive. Nearly all were sensitive to Cf. A lawn of Cflt-infected cells did not form plaques with Cflt but it did with Cf. Thus, Cflt infection produces immunity
308
KU0
FIG. 1. Electron
micrograph
of phage
Cflt.
ET
The phages
to Cflt, but not to Cf, in roughly 70% of the infected cells. Immunity of Cflt-infected cells to superinfection with Cflt is correlated with another property, the yield of phage from the infected cells, shown in Table 1. Cfltinfected cells can be divided into high yielders, pro123456
FIG. 2. Determination of the molecular size of Cflt DNA. Cflt RF DNA was digested with BarnHI and Hincll, respectively. Digested DNA fragments were electrophoresed horizontally for 12 hr in a 1% agarose gel at 40 V in TBE buffer. X DNA digested with HindIll was used as size marker. Cf RF DNA digested with same enzyme was used for comparison. Lane 1, X DNA digested with HindIll and EcoRI; lane 2, Cflt RF DNA digested with BarnHI; lane 3, Cf RF DNA digested with BarnHI; lane 4. Cflt RF DNA digested with Hincll; lane 5, Cf RF DNA digested with Hincll; lane 6. X DNA digested with HindIll.
AL.
were
stained
with
2% phosphotungstic
acid
ducing a phage titer over 10’ PFU/ml, and low yielders, producing a phage titer below 1O7 PFU/ml. Cflt-immune cells were predominantly low yielders (959/o) while sensitive cells were predominantly high yielders (91%). Stability of immunity
and phage yield
These properties were tested by transferring 30 colonies of each type through five passages, isolating single colonies at each passage. Among immune colonies, the original properties of immunity and low phage yield were completely retained. Among sensitive col-
FIG. 3. Plaque
morphology
of Cf (left) and Cflt (right)
LYSOGENIC
CYCLE
OF
X. campesrris
TABLE
PHAGE
309
1
IMMUNITY AND PHAGE YIELD IN Cflt-INFECTED
CELLS
Immunity
Immune
Sensitive
to Cflt
to Cflt
Phage
No. of cells obtained
%
141
70
Low phage High phage
59
30
Low phage High phage
yield No. of cells obtained
%
yielder yielder
131 8
95 5
yielder yielder
6 53
9 91
onies, 28 retained their original properties while 2 became free of phage.
original properties, phage yielders.
Effect of lysogeny on growth
Induction
The lysogenic cells were grown in LB medium and at different times bacterial growth and phage yield were measured (Fig. 4). The lysogen grew at the same rate as uninfected bacteria and the phage yield did not increase.
Phage production in lysogenized cell cultures was always between 1O6 and 10’ PFU/ml. However, when these cultures were grown and transferred several times, the phage titer increased to 10g-lO’o PFU/ml. We suspected this was due to the conversion of low yielders into high yielders. To test this possibility, a single lysogen was grown in LB and samples were taken at various times to determine the proportion of low phage yielders, high phage yielders, and cured cells. The results are summarized in Table 2. Three independent experiments gave vet-y similar results. Over a 48-hr period, 21% of the low phage yielders were converted to high phage yielders and 8% were cured. The cured cells were sensitive to Cflt infection.
The persistence
of prophage
in Cflt-infected
cells
The ability of immune cells to produce phage, after growth in LB containing antiserum, was determined. Figure 5 shows that the antiserum effectively killed all phages released into the medium, preventing reinfection. After growth for 24 generations in antiserum-containing medium, 100 individual colonies were tested for immunity and phage yield. Ninety-five retained their
I
and curing of Cflt lysogens
I
1
I
I
I
I
I
I
2
4
6
6
10
12
14
16
TIME
3 were cured, and 2 were high
1
I 20
(h)
FIG. 4. Growth of Cflt-lysogenized cells and phage production in LB medium at 30” with shaking. phage titer were measured as described under Materials and Methods. 0, Growth of Cflt lysogenized A, Phage production in a lysogen culture.
The number of bacterial cells and the cells. 0, Growth of uninfected bacteria.
310
KU0 IO’
= 5 g
ET AL.
tion of Cflt DNA into host DNA occurs, the DNAs from Cflt-infected cells, Cf-infected cells, noninfected host cells, and Cflt RF were isolated and digested with BarnHI, HindIll, and Pvull, respectively. The resulting fragments were fractionated by agarose gel electrophoresis. The DNA fragments in the gel were then denatured in situ, transferred to a hybridization membrane, and hybridized to Cflt RF DNA that had been labeled with [32P]lTP by nick translation (Rigby et a/., 1977). The location of viral sequences among the fragments of bacterial chromosome DNA was determined by autoradiography. The results in Fig. 6 show that Cflt DNA hybridized only with chromosomal DNA isolated from Cflt-infected cells, not with DNA from uninfected host cells. Since the only band detected was in the position of host chromosome DNA it appears that there was no free form of phage DNA in the Cflt-infected cell (Fig. 6, lanes 1, 2). Two bands from Cflt RF DNA were detected; one is supercoiled (RF I) and another, open circular (RF II). When Cflt RF was isolated from newly infected cells, both forms of RF were usually found. When RF DNA was cut with HindIll, two fragments of 5.7 and 1.6 kb were produced. When the DNA from Cflt-infected cells was digested with the same enzymes, three fragments of 4.6, 2.9, and 1.6 kb were obtained. The 5.7-kb fragment was replaced by two new fragments (Fig. 6, lanes 5, 10). To confirm this observation two other restriction enzymes, Pvull and BarnHI, were tested with similar results. m/ull cut Cflt RF into two fragments of 5.2 and 2.4 kb, but cleaved Cflt-infected cell DNA into three fragments of 5.2, 2.3, and 1.3 kb. The 5.2-kb fragment was preserved and the 2.4-kb fragment was replaced by two new fragments of 2.3 and 1.3 kb (Fig. 6, lanes 6, 11). BarnHI has one site on RF DNA. When the DNA isolated from Cflt-infected cells was cleaved by the same enzyme, the Cflt RF DNA fragment was missing, and two bands of 15.6 and 14.4 kb were detected. On this gel the two bands are so close that only one thicker band was observed (Fig. 6, lanes 4, 9). These data provide the
r
lo5
10'
a tw F w
io3
F! lo*
10'
loo 5
13
19
24
29
36
42
46
TIME(h) FIG. 5. Persistance of prophage in the presence of Cflt antiserum. Lysogen was grown in 8 ml LB broth to which previously 4 ml of 10-l antiserum against Cflt had been added. For control the antiserum was omitted. The cultures were incubated at 30”. At different time intervals, 1 ml of cultured cells was taken to be centrifuged at 10,000 g for 20 min. The phage titer in supernatant was determined. 0, Phage titer in lysogenic culture with antiserum. 0, Phage titer in lysogenic culture without antiserum.
These figures, 21% high yielders and 8% cured cells, are in good agreement with the proportion of these classes formed following Cflt infection (Table 1). Physical evidence of the integration into host chromosome DNA
of Cflt DNA
The stability of phage yield and immunity to superinfection suggests that each daughter cell inherited the phage genome faithfully during cell division. Cells may have transmitted the phage DNA either as a plasmid (Ikeda and Tomizawa, 1965) or as an integrated prophage (Hershey, 1971). To determine whether integraTABLE
2
INDUCTION AND CURING OF Cflt LYSOGEN UNDER NATURAL CULTIVATION Incubation Types
of cells
(%I
0
Phage free cell High phage yielder Low phage yielder
Note. A pure lysogen samples medium.
from this culture The phage yield
8 0 0
100 was
0 0 100
time
CONDITIONS
(hr)
16
24
32
0 0 100
1
3
3 96
10
87
40
48
4 12 84
8 21 71
selected by single colony isolation, transferred to LB medium, and grown at 30” with shaking. At different times were spread on an LB agar plate. After colony formation, 200 colonies were tested by transfer into 1 ml of LB in each culture was determined as described under Materials and Methods.
LYSOGENIC
1 2 3 4 5 6 7 6 9 10 11
CYCLE
12
FIG. 6. Integration of Cflt DNA into host chromosome. The DNAs from Cflt RF, Cflt lysogen, Cf infected cells, and noninfected host cells were digested with BarnHI, Hindlll, and Pvull. All digests were electrophoresed horizontally for 16 hr in a 0.8% agarose gel at 30 V in TEE buffer, blotted, and hybridized with nick-translated 32P-labeled Cflt RF DNA. X DNA digested with HindIll was used as size markers. The amount of DNA used in each sample was 0.5 rg for X DNA, 0.2 pg for Cflt RF DNA and 20 pg for lysogen DNA, Cf infected cells and host cells. Lane 1, DNA from uninfected host cells; lane 2, undigested DNA from Cflt lysogen; lane 3, DNA from uninfected host cells digested with BarnHI; lane 4, DNA from Cflt lysogen digested with BarnHI; lane 5, DNA from Cflt lysogen digested with HindIll; lane 6, DNA from Cflt lysogen digested with Pvull; lane 7, DNA from Cf infected cells; lane 8, Cflt RF DNA; lane 9, Cflt RF DNA digested with BarnHI; lane 10, Cflt RF DNA digested with HindIll; lane 11, Cflt RF DNA digested with h/VII; lane 12, X DNA digested with HindIll.
OF X.
campestris
PHAGE
311
growth of infected cells, and has a low phage yield, whereas Cf forms clear plaques, slows the growth of infected cells, and has a high yield. Moreover, Cflt infection results in the formation of lysogens immune to superinfection by Cflt, while infection by Cf does not produce lysogens. To date, lysogens are known to be produced only by viruses containing double-stranded DNA. This is the first report of lysogenization by a virus containing circular single-stranded DNA. Cflt-infected bacteria can be divided into two types on the basis of immunity and phage yield. In a typical infection, about 70% of the infected cells become immune, low yielders while 30% are sensitive, high yielders. Single colony isolation of infected cells led to the conclusion that each type was relatively stable, except that after repeated transfer, the lysogenic type of cell could be converted to the sensitive high yielder (20%) or cured (8%). The very low production of phage by lysogens was confirmed by our inability to find Cflt RF DNA in lysogenized cells. We do not believe that the conversion of immune, low yielders to sensitive, high yielders is an artifact due to contamination of the cultures. If this were true, we should have detected the contaminating cells at an early stage, since both lysogens and cured cells grow at the same rate (Table 2). We have tried to induce or to cure lysogens with UV light, heat, ethidium bromide, and acridine orange, without success. Similar failures have been observed 12345676
evidence that the Cflt DNA was integrated into the host DNA. To study the frequency and the pattern of Cflt DNA integration, 15 infected colonies were randomly picked, and the DNAs from these isolates were digested with BarnHI and hybridized with 32P-labeled Cflt RF DNA. The result is shown in Fig. 7. All Cflt lysogens contained integrated phage DNA. Two bands were detected in all Cflt lysogens. Since the position of the two samesized bands was always identical, it means that one copy of Cflt DNA is integrated at same site on host DNA. DISCUSSION
Comparison of virion morphology, serology, and the size and form of native viral DNA showed phage Cflt to be similar to Cf, a previously characterized phage containing single-stranded circular DNA (Dai et a/., 1980). However, the two phages could be distinguished: Cflt forms turbid plaques, does not affect the
FIG. 7. The integration pattern of viral DNA in Cflt lysogens. DNAs were isolated from Cflt lysogens and digested with BarnHI. The conditions for gel electrophoresis and Southern hybridization were described as in the legend of Fig. 6. Lane 1, Cflt RF DNA; lane 2, Cflt RF DNA digested with BarnHI; lane 3, DNA from Cflt lysogen P41, digested with BarnHI; lane 4, DNA from Cflt lysogen P37, digested with BarnHI; lane 5, DNA from Cflt lysogen P31, digested with BarnHI; lane 6, ONA from Cflt lysogen d4, digested with BarnHI; lane 7, DNA from Cflt lysogen 1 1, digested with BarnHI; lane 8, X DNA digested with HindIll.
312
KU0
in other lysogenic systems (Barksdale and Arden, 1974; Orndorff et al., 1983). Growth of Cflt lysogens through viral antiserum proves that both immunity and ability to produce phage are stable properties of the lysogens, not due to reinfection. Two kinds of stable lysogens of bacterial viruses are known. In one, the phage DNA is integrated into the bacterial chromosome (Hershey, 1971). In the other, the phage DNA is a plasmid whose replication is coordinated with that of the chromosome (Ikeda and Tomizawa, 1965). Our Southern blots demonstrate that Cflt DNA is integrated into the host chromosome. The band patterns produced by cutting individual lysogens with different restriction enzymes (Figs. 6 and 7) allow us to conclude that each lysogenic cell has only one integration site and that the site is at the same chromosomal location in each lysogen. ACKNOWLEDGMENTS We thank Wen C. Wu for providing us X. campestris pv. citri strain XW47-11. The work was supported by a grant from National Science Council, Republic of China.
REFERENCES ADAMS, M. H. (1959). Antigenic properties. In “Bacteriophages,” pp. 97-l 17. Wiley Interscience, New York. BAAS, P. D. (1985). DNA replication of single-stranded Escherichia co/i DNA phages. Biochim. Biophys. Acta 825, 1 1 l-l 39. BARKSDALE, L., and ARDEN, S. B. (1974). Persisting bacteriophage infections, lysogeny, and phage conversions. Annu. Rev. Microbial. 28,265-229. BIRNBOIM, H. C., and DOLY, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513-l 523.
ET AL. BUKHARI, A. I., and METLAY, M. (1973). Genetic mapping of prophage Mu. Virology 54, 109-l 16. CLEWELL, D. B. (1972). Nature of Col El plasmid replication in the presence of chloramphenicol. J. Bacterial, 110, 667-676. DAI, H., CHIANG, K. S., and Kuo, T. T. (1980). Characterization of a new filamentous phage Cf from Xanthomonas citri. /. Gen. Viral. 46,277-289. DAI, H., TSAY, S.-H., Kuo, T.-T., LIN, Y.-H., and Wu, W. C. (1987). Neolysogenization of Xanthomonas campestris pv. citri infected with filamentous phage Cfl6. Virology 156, 313-320. DENHARDT, D. T., Rnv, D. S., and DRESSLER, D. (1978). “Single-Stranded DNA Phages.” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. EISENSTARK, A. (1967). Bacteriophage techniques. /n “Methods in Virology” (K. Maramorosch and H. Koprowaki, Eds.), Vol. 1, pp. 449-524. Academic Press, New York. HERSHEY, A. D. (1971). “The Bacteriophage Lambda,” p, 792. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. HSU, Y. C. (1968). Propagation or elimination of viral infection in carrier cells. Bacteria/ Rev. 32, 387-391, IKEDA, H., and TOMIZAWA. J. (1965). Transducing fragments in generalized transduction by phage P1.J. Mol. Biol. 14, 85-109. MARVIN, D. A., and HOHN, B. (1969). Filamentous bacterial viruses. Bacterial. Rev. 33, 172-209. MERRIAN, V. (1977). Stability of the carrier state in bacteriophage M 13. infected cells. J. Viral. 21, 880-888. ORNDORFF, P., STELLWAG. E., STARICH, T.. DWORKIN, M., and ZISSLER, J. (1983). Genetic and physical characterization of lysogeny by bacteriophage MX8 in Myxococcus xanthus. J. Bacterial. 154, 772779. RIGBY, P. W. J., DIECKMAN, M., RHODES, C., and BERG, P. (1977). Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 113, 237251. SOUTHERN, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. f. Mol. Biol. 98, 501517. WILLIAMS, C. A., and CHASE, M. W. (Eds.) (1967). “Methods in Immunology and Immunochemistry,” Vol. 1, pp. 15-l 20. Academic Press, New York. YANG, M. K., and Kuo. T. T. (1984). A physical map of the filamentous bacteriophage Cf genome. 1. Gen. Viral. 65, 1175-l 181.