FEMS Microbiology Letters 192 (2000) 119^124
www.fems-microbiology.org
The life cycles of the temperate lactococcal bacteriophage PLC3
monitored by a quantitative PCR method
Merete Lunde *, Janet Martha Blatny, Fiona Kaper 1 , Ingolf F. Nes, Dag Lillehaug
2
Laboratory of Microbial Gene Technology, Department of Chemistry and Biotechnology, Agricultural University of Norway, P.O. Box 5051,
N-1432 Aas, Norway
Received 30 June 2000; received in revised form 4 September 2000; accepted 10 September 2000
Abstract
We present here a new and general approach for monitoring the life cycles of temperate bacteriophages which establish lysogeny by
inserting their genomes site-specifically into the bacterial host chromosome. The method is based on quantitative amplification of specific
DNA sites involved in various cut-and-join events during the life cycles of the phages (i.e. the cos, attP, attB, attL and attR sites) with the
use of sequence-specific primers. By comparing the amounts of these specific DNA sites at different intervals, we were able to follow the
development of the lytic and lysogenic life cycles of the temperate lactococcal bacteriophage PLC3 after infection of its bacterial host
Lactococcus lactis ssp. cremoris IMN-C18. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
All rights reserved.
Keywords : Lactococcus lactis; Bacteriophage PLC3; Quantitative polymerase chain reaction; Monitoring the life cycles of temperate phages
1. Introduction
Bacteriophages impose a threat to the fermentation industry because of their ability to infect lactic acid bacteria,
particularly Lactococcus lactis, used in starter cultures and
thereby distort a variety of food fermentation processes
[1]. The virulent bacteriophages have been much more
thoroughly studied than temperate bacteriophages because
they are apparently more abundant. However, many
strains of L. lactis are found to be lysogenic, carrying inducible prophages [2]. The signi¢cance of temperate bacteriophages in negatively in£uencing a fermentation process is not known. Major reasons for this limited insight is
probably due to the lack of suitable methods to observe
lysogeny and to study the molecular biology of the life
cycles of temperate phages.
* Corresponding author. Tel. : +47 (64) 94 94 65;
Fax: +47 (64) 94 14 65; E-mail : [email protected]
1
Present address: Department of Radiation Oncology, Cancer Biology
Research Laboratory, Stanford Medical Center, CA 94305-5468, USA.
2
Present address: Norwegian Food Control Authority, P.O. Box 8187,
N-0034 Oslo, Norway.
A temperate bacteriophage decides early in the infection
cycle to enter either the lytic or the lysogenic pathway.
After the phage DNA has entered the host cell, the genome of V-like phages converts from a linear to a covalently closed circular (ccc) DNA molecule by annealing
two single-cos sites to establish one single double-cos site
in the circular phage genome (Fig. 1). The cccDNA genome is the substrate for the replication during lytic
growth as well as for integration of the prophage within
the bacterial chromosome during establishment of lysogeny. In the latter event, the phage inserts its genome into
the bacterial chromosome by site-speci¢c recombination
between the phage (attP) and the bacterial (attB) attachment sites resulting in two new attachment sites (attL and
attR) at the phage^host DNA junctions. Following induction of the lytic pathway and excision of the integrated
prophage, attL and attR recombine to re-establish the
attP and attB sites in the phage and host chromosomes,
respectively [3].
In general, the lysogenic bacteria have been studied by
analyzing the immunity to superinfection and by phage
induction during bacterial growth [4], while the lytic process ending in cell lysis and phage release usually has been
observed by cell turbidity measurements and plaque assays
[5]. Whereas the techniques used in these analyses are only
able to determine the end point of the phage life cycle, we
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 4 2 1 - 3
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M. Lunde et al. / FEMS Microbiology Letters 192 (2000) 119^124
2.2. DNA techniques
Fig. 1. Schematic presentation of speci¢c DNA regions involved in various cut-and-join events during the life cycles of the lambda-like phages,
including phage PLC3. The thin line, thick line, circle and double circles
indicate phage DNA, chromosomal DNA of the host, single-stranded
cohesive ends on the linear form of the phage (single-cos) and ligated
cohesive ends on the circularized phage genome (double-cos), respectively. Attachment sites for site-speci¢c integration between attP (phage)
and attB (bacteria) generating the attR and attL sites in the phage^host
junction are indicated as open boxes and closed boxes for phage DNA
and bacteria DNA, respectively.
have monitored the development of the lytic and lysogenic
pathways of the temperate lactococcal PLC3 after infecting its host L. lactis IMN-C18 by the use of a method
presented in this report. The method is based on the use
of quantitative competitive polymerase chain reaction
(PCR) [6] directed towards the attB, attP, attL, attR and
cos sites of PLC3 (Fig. 1), and comparison of the level of
these sites at di¡erent points of time after infection. The
genetic elements required for the integration of PLC3 into
the chromosome of its bacterial host and the cos region of
PLC3 have previously been mapped and sequenced [7,8].
To our knowledge, this is the ¢rst report in which the
relative amounts of recombining DNA sites have been
used as a target to study temperate phage biology.
If nothing else is stated, the isolation and manipulation
of DNA were performed using standard techniques [9].
DNA fragments used to construct the control templates
were puri¢ed by agarose gel electrophoresis and eluted
from the gel by use of the MerMaid kit (Bio 101). Construction of control templates were based on the site-speci¢c mutagenesis technique of Higuchi et al. [10] (Fig. 2).
Template DNA for the quantitative PCR assay was obtained by a freeze^thaw technique as described [11]. PCR
products were puri¢ed by the use of Qiaquick (Qiagen).
The cos- and attR-control templates were ampli¢ed from
L. lactis IMN-C3 DNA; attB- and chr-control templates
were ampli¢ed from L. lactis IMN-C18 DNA and the
attP-control template was ampli¢ed from PLC3 DNA.
Standard PCR was performed using the Taq DNA polymerase kit from Advanced Biotechnologies; quantitative
competitive (QC)-PCR was performed by use of the GeneAmp kit and AmpliTaq Gold DNA polymerase (PerkinElmer), in the Perkin-Elmer 9600 thermal cycler. In the
QC-PCR reaction, one primer in each primer-set was labeled with 5Pcarboxy£uorescein (6-FAM). The oligonucleotide primers used in the ampli¢cation reactions are
shown in Tables 1 and 2.
2.3. QC-PCR
All PCR mixtures, except those containing the attR template, were kept at 95³C for 12 min prior to ampli¢cation
to activate the AmpliTaq Gold DNA polymerase and ampli¢ed for 20^25 cycles. Due to the low level of attR DNA
in the infected bacterial cultures observed in preliminary
2. Materials and methods
2.1. Bacterial strains, growth media and phage
The PLC3 indicator strain L. lactis ssp. cremoris IMNC18 and the PLC3-lysogenic strain L. lactis ssp. cremoris
IMN-C3 were grown at 30³C in M17 broth (Oxoid) containing 5 g l31 glucose instead of lactose. For propagation
of PLC3, 5 mmol l31 of CaCl2 was added to the M17
medium. Stocks of PLC3 phage particles were prepared
as described previously [7].
Fig. 2. Construction of control templates. In step 1a and 1b, two PCR
products are generated by the use of primers (Tables 1 and 2) containing one base exchange (indicated by x) resulting in the construction of a
unique restriction enzyme site (indicated by b). Primers for ampli¢cation of the various fragments are indicated by arrows. The two PCR
products, which are homologous in the mutated region, were mixed,
and the £anking primers were used in the second PCR reaction (step 2).
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M. Lunde et al. / FEMS Microbiology Letters 192 (2000) 119^124
121
Table 1
Nucleotide sequence of PCR primers used in the construction of control templates
Ampli¢ed
regiona
Primer sequenceb
Step in
Fig. 2
Base
transition
Annealing
temperature (³C)
attP
5P-CGGGATCCTGTCTGACGGCTGGGTAATGT-3P
5P-GTTGTAAGAAAATTATAACTGCAGTAGCAGCTC-3P
5P-GAGCTGCTACTGCAGTTATAATTTTCTTACAAC-3P
5P-AATATATTCGGGATGATTGTGGGA-3P
5P-CGGGATCCTGTCTGACGGCTGGGTAATGT-3P
5P-AATATATTCGGGATGATTGTGGGA-3P
5P-CGGGATCCTGTCTGACGGCTGGGTAATGT-3P
5P-GTTGTAAGAAAATTATAACTGCAGTAGCAGCTC-3P
5P-GAGCTGCTACTGCAGTTATAATTTTCTTACAAC-3P
5P-CTAAATAATAAACTAAAAATTTGTGAAAAAATTAAAATG-3P
5P-CGGGATCCTGTCTGACGGCTGGGTAATGT-3P
5P-CTAAATAATAAACTAAAAATTTGTGAAAAAATTAAAATG-3P
5P-TGGGGTAGATGCTGCCAC-3P
5P-GAATAACAAGGTTTGGAATTCTTTAGTTAAATC-3P
5P-GATTTAACTAAAGAATTCCAAACCTTGTTATTC-3P
5P-CTAAATAATAAACTAAAAATTTGTGAAAAAATTAAAATG-3P
5P-TGGGGTAGATGCTGCCAC-3P
5-CTAAATAATAAACTAAAAATTTGTGAAAAAATTAAAATG-3P
5P-TGATTCCTGTTGCATTGACCGC-3P
5P-GGATACCTGAGATTTAAGCTTTCTGTTCATAAACG-3P
5P-CGTTTATGAACAGAAAGCTTAAATCTCAGGTATCC-3P
5P-GCCAGAAGTCTGACAAGCTATTCAATGTG-3P
5P-TGATTCCTGTTGCATTGACCGC-3P
5P-GCCAGAAGTCTGACAAGCTATTCAATGTG-3P
5P-TTGCCACAATACTATCGACTTTTC-3P
5P-GAAGCGCACGTCCAAGCTTTCCGACTCCAATC-3P
5P-GATTGGAGTCGGAAAGCTTGGACGTGCGCTTC-3P
5P-CATGAAGAATAACAAGGTTTGCAATTC-3P
5P-TTGCCACAATACTATCGACTTTTC-3P
5P-CATGAAGAATAACAAGGTTTGCAATTC-3P
1a
C
52
1316
1b
G
52
352
57
1568
attP
attP
attR
attR
attR
attB
attB
attB
cos
cos
cos
chr
chr
chr
2
Size of fragments
(bp)
1a
C
52
1316
1b
G
52
252
59
1468
2
1a
C
52
493
1b
G
52
177
54
637
2
1a
A
58
370
1b
T
58
303
62
638
2
1a
G
52
256
1b
C
52
358
54
582
2
a
cos = double-cos.
The PLC3 speci¢c primers are based on the PLC3 sequence previously presented [7,8]. The PLC3 attB speci¢c primers are based on the PLC3 attB sequence (unpublished).
b
studies, attR DNA was ampli¢ed for 40 cycles, resulting in
a step-wise activation of the AmpliTaq Gold DNA polymerase. The ampli¢cation pro¢le was performed by 70 s at
94³C, 30 s at the appropriate annealing temperature (Table 2), followed by 3 min at 72³C.
2.4. Quantitative analyses of PCR products
The appropriate amounts of the puri¢ed PCR products
for restriction analyses were determined by loading 3 Wl of
a mix containing 1 Wl puri¢ed PCR products and 5 Wl
loading bu¡er (1:5, formamide and non£uorescent gel
loading bu¡er) on a 4.2% polyacrylamide gel in 1UTBE
bu¡er (pH 8.3). The £uorescent signals were analyzed by
the use of an automatic DNA sequencer during 1 h (750 V
at 51³C) (ABI Prism 377, Perkin-Elmer). The total PCR
mixtures were digested with the corresponding restriction
endonuclease (Table 2) and then desalted by di¡usion for
20 min (0.05 mm Millipore ¢lter and 0.1UTE bu¡er, pH
8.0). The desalted digested PCR products were mixed with
loading bu¡er and the samples were denatured. Fragments
of the control and target templates were separated by elec-
trophoresis as described above. The GeneScan 500 TAMRA-labeled DNA from Perkin-Elmer was used as a molecular mass standard.
Data were collected and processed using the GeneScan
analysis software (version 1.1) according to the manufacturer's guidelines. The amount of £uorescence incorporated in each fragment was analyzed and the ratios of
the control and target template were determined allowing
the unknown amount of target template in each sample to
be estimated.
3. Results
The temperate lactococcal bacteriophage PLC3 and its
bacterial host L. lactis IMN-C18 were used as a model
system to study the infection cycle of temperate bacteriophages by a new approach based on the use of QC-PCR.
An exponentially growing bacterial culture was infected
with the phage (multiplicity of infection of 10), and the
level of double-cos sites and attB, attR and attP attachment sites were measured at regular intervals. A speci¢c
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M. Lunde et al. / FEMS Microbiology Letters 192 (2000) 119^124
Table 2
Nucleotide sequence of primers used in the competitive PCR assay
Ampli¢ed
region
Primer sequencea
Restriction
enzyme
Annealing
temperature (³C)
Size of fragments,
target/control template (bp)
attP
*5P-TTCAAAACGCCTAGGACACGC-3P
5P-ACATAATGATATGTTGTAATTTTGTTGTACGAG-3P
*5P-TTCAAAACGCCTAGGACACGC-3P
5P-TGTGAAAAAATAAAATGATGATGGC-3P
*5P-TCTTAACTTTGCACCTATTCGCTTG-3P
5P-TGTGAAAAAATAAAATGATGATGGC-3P
5P-GCTTAATGCTTGTGGAATTGTAATCCC-3P
*5P-CCCCCTCCCTCTGGGTCAGC-3P
*5P-AATTAGGACGAGGCTACGG-3P
5P-GATTTCCAGAAATATCGTAGGCC-3P
PstI
58
344/111
PstI
58
328/111
EcoRI
58
214/69
HindIII
60
219/118
HindIII
58
193/106
attR
attB
cos
chr
a
Labeled with 6-FAM at the 5P terminus.
bacterial chromosomal sequence designated chr, located
adjacent to the attB-core region, was included in the assay
as a control for any possible di¡erences in DNA isolation
e¤ciency and DNA ampli¢cability between the di¡erent
DNA samples. The results presented in Fig. 3 showed that
for chr, the target and control templates were ampli¢ed
with the same e¤ciency over a two exponential range during competitive ampli¢cation. The same level of accuracy
was obtained with the attB, attR, attP and double-cos
DNA regions (data not shown), thus establishing that all
target templates can be measured quantitatively.
By introducing PLC3 phage DNA into the L. lactis host
cells by infection, di¡erent events such as circularization,
concatemerization, site-speci¢c integration and excision of
the phage genome may be monitored due to changes in the
levels of the speci¢c double-cos sites and the attachment
sites. Fig. 4 shows the changes in the levels of these sites
from the time of infection until the end of the lytic cycle.
The double-cos sites were detected 1 min after the infection and probably represent the ¢rst circularization of the
linear phage genome within the bacterial cells. The level of
the attP sites, which is present both in the linear and in the
circular form of the phage genomes, was detected at a
constant level of approximately 10 per cell during the ¢rst
30 min, whereas the double-cos sites increased from 0.1 to
10 per cell during this interval. The increase of double-cos
sites most likely re£ects the rate of penetration and circulation of the linear phage genome into its cccDNA form.
The approximately six-fold increase in the level of attP
during the next 20 min probably represents the period
and level of phage replication, resulting in approximately
60 phage genomes per cell at the end of the lytic cycle. The
results from the QC-PCR assay con¢rmed the previous
¢ndings from a previously performed one-step growth experiment of PLC3 where the burst size was estimated to
approximately 50 phages and the latent period was about
Fig. 3. Ampli¢cation e¤ciency and quanti¢cation of target template.
Competitive PCR was performed with a dilution series of chr control
template, in which each vial contained an equal unknown amount of
chr target template. The concentration of the control template ranged
from 0.0146 to 14.64U10318 mol Wl31 . Data in the ¢gure are plotted as
exponential control/target DNA PCR product versus exponential control
DNA. The regression line with an R2 value of 0.99 is given by
y = 0.962x+0.0527, in which y is the equivalence point between the control and target ratio. Substituting y with 1 in the formula, gives
x = 0.885 10318 mol Wl31 , which is the amount of the chr target DNA
added to the reaction.
Fig. 4. The use of QC-PCR for analyzing the infection process of
PLC3. The chromosomal marker chr (F) represents the total amount of
bacterial cells. The level of double-cos regions (a) indicates the number
of circularized and integrated phage genomes as well as phages in concatemers. The attB region (8) represents the number of cells without
prophage and the attP region (7) represents the number of unintegrated phage genomes. The level of attR (^) indicates the level of lysogenic cells. The various regions were ampli¢ed from total DNA extracts
obtained from an exponentially growing culture of L. lactis IMN-C18
infected with PLC3 at a multiplicity of infection of 10. Samples of 3 ml
were withdrawn at the di¡erent points of time indicated in the ¢gure.
FEMSLE 9642 12-10-00
M. Lunde et al. / FEMS Microbiology Letters 192 (2000) 119^124
60 min, including an eclipse period of about 40^50 min
and a maturation period of 5^10 min [7].
In addition to the studies of the lytic cycle above, the
lysogenization process of PLC3 was analyzed. In order to
establish stable lysogeny it has been found that only a
single copy of the phage genome is inserted into the host
chromosome by a site-speci¢c recombination event
through attP and a preferentially attB site [7,8]. Based
on this ¢nding, the frequency of spontaneous formation
of lysogenic L. lactis IMN-C18 was measured by quantifying the level of the attR attachment site at regular intervals after infection. The attR site was ¢rst detected at a
level of 1 per 10 000 cells 20 min after infection. During
the following 40 min the amount of attR sites increased to
a constant level of approximately one out of 1000 cells.
This data strongly suggested that lysogeny is established in
approximately one out of 1000 infected bacterial cells. In a
preliminary experiment the increase in the level of the attL
site was monitored at the same level as for attR (data not
shown). It was not possible to detect the corresponding
decrease in the low level of attB signi¢cantly.
4. Discussion
In this report an assay based on the use of QC-PCR has
been developed and used to study the infection of the
temperate lactococcal phage PLC3 in L. lactis ssp. cremoris IMN-C18. By quantifying the di¡erent cut-and-join
sites involved in the various stages in the life cycles of
this phage, we showed that it was possible to distinguish
between di¡erent forms of the phage genome as it appeared during the infection process (Fig. 1). Thus, the lytic
and lysogenic pathways were followed by analyzing total
DNA obtained from the infected bacterial culture at different points of time after infection.
Due to the exponential nature of the PCR reaction and
the fact that a small £uctuation in the ampli¢cation e¤ciency can a¡ect the yield of PCR products, several requirements have to be ful¢lled in order to perform quantitative analysis by PCR. Comprehensive testing and
controls are required to generate reproducible and statistically valid results. Consequently, we have used both an
external DNA control template, which acts as an `in-tube'
reference for the ampli¢cation, and an internal chromosomal DNA control marker in order to compare the amount
of the various PCR products in DNA extracts sampled at
di¡erent points of time after infection. It is of crucial
importance in QC-PCR analysis that the two competitive
DNA templates are to be ampli¢ed with equal e¤ciency,
such that the ratio of the external control and target DNA
PCR products is kept constant during the ampli¢cation
reaction [12]. This allows quanti¢cation of the target region, since the amount of external control template added
to the reaction is known. The slope of the line in the
exponential plot (Fig. 3) is close to unity, showing that
123
the ampli¢cation products can be accurately measured
by this method [13].
To quantify low concentrations of the DNA target, such
as attR in the present assay, at least 40 PCR cycles were
required by the method used. The amount of attR was
therefore at the lower limit of the sensitivity of the assay,
since this high number of cycles may increase the possibility of heteroduplex formation [6], which can further
result in incomplete digestion of the PCR mixture leading
to an overestimation of the target template. To ensure
complete digestion of the control template, a separate
vial containing only control template was used as a control since the digestion step is of major importance in the
QC-PCR assay.
Quantitative PCR has proven to be a powerful tool to
study the infection cycle of the temperate lactococcal bacteriophage PLC3. At present, the closed-tube real-time detection PCR ampli¢cation by the ABI Prism 7700 Sequence Detection System (Perkin-Elmer) is being used
for quantitative analysis in our phages studies. No postPCR sample handling is needed in this system, the dynamic range of the quantitative analysis is about 6 exponential
units, and the time needed to perform the analysis is less
than other quantitative PCR methodologies. In our laboratory we are currently studying the in£uence of environmental factors on the phage decision to develop lytically
or lysogenically. In addition, studies of the importance of
host- and phage-encoded genes that might be involved in
the process of lysogenization and maintenance of PLC3
lysogeny will be carried out.
We believe that the method presented here is a valuable
tool in future studies of temperate bacteriophage biology.
These studies are needed in order to elucidate the in£uence
of temperate bacteriophages in fermentation processes.
Acknowledgements
This work was supported by grants from the European
Union Biotechnology programme (Contract BIO4-CT960402) and the Norwegian Research Council.
References
[1] McKay, L.L. and Baldwin, K.A. (1990) Applications for biotechnology: present and future improvements an lactic acid bacteria. FEMS
Microbiol. Rev. 87, 3^14.
[2] Davidson, B.E., Powell, I.B. and Hillier, A.J. (1990) Temperate bacteriophages and lysogeny in lactic acid bacteria. FEMS Microbiol.
Rev. 87, 79^90.
[3] Landy, A. (1989) Dynamic, structural, and regulatory aspects of V
site speci¢c integration. Annu. Rev. Biochem. 8, 913^949.
[4] Ptashne, M., 1992. A Genetic Switch: Phage V and Higher Organisms. Cell Press and Blackwell Scienti¢c Publications, Cambridge,
MA.
[5] Young, R. (1992) Bacteriophage lysis : mechanism and regulation.
Microbiol. Rev. 56, 430^481.
FEMSLE 9642 12-10-00
124
M. Lunde et al. / FEMS Microbiology Letters 192 (2000) 119^124
[6] Gilliland, G., Perrin, S., Blanchard, K. and Bunn, H.F. (1990) Analysis of cytokine mRNA and DNA: detection and quantitation by
competitive polymerase chain reaction. Proc. Natl. Acad. Sci. USA
87, 2725^2729.
[7] Lillehaug, D., Lindqvist, B. and Birkeland, N.K. (1991) Characterization of PLC3, a Lactococcus lactis ssp. cremoris temperate bacteriophage with cohesive single-stranded DNA ends. Appl. Environ.
Microbiol. 57, 3206^3211.
[8] Lillehaug, D. and Birkeland, N.K. (1993) Characterization of genetic
elements required for site-speci¢c integration of the temperate lactococcal bacteriophage PLC3 and construction of integration-negative
PLC3 mutants. J. Bacteriol. 175, 1745^1755.
[9] Sambrock, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning : a
Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory, Cold
Spring Harbor, NY.
[10] Higuchi, R., Krummel, B. and Saiki, R.K. (1988) A general method
of in vitro preparation and speci¢c mutagenesis of DNA fragments:
study of protein and DNA interactions. Nucleic Acids Res. 16, 7351^
7367.
[11] Head, I.M., Hiorns, W.D., Embley, T.M., McCarthy, A.J. and Saunders, J.R. (1993) The phylogeny of autotropic ammonia-oxidizing
bacteria as determined by analysis of 16S ribosomal RNA gene sequences. J. Gen. Microbiol. 139, 1147^1153.
[12] Siebert, P.D. and Larrick, J.W. (1992) Competitive PCR. Nature 359,
557^558.
[13] Raeymaekers, L. (1993) Quantitative PCR: theoretical considerations
with practical implications. Anal. Biochem. 214, 582^585.
FEMSLE 9642 12-10-00