Previous Article | Next Article 
Journal of Bacteriology, December 1999, p. 7291-7297, Vol. 181, No. 23
Department of Microbiology, Technical
University of Denmark, DK-2800 Lyngby, Denmark
Received 1 June 1999/Accepted 17 September 1999
In this work, the phage-encoded proteins involved in site-specific
excision of the prophage genome of the temperate lactococcal bacteriophage TP901-1 were identified. The phage integrase is required
for the process, and a low but significant frequency of excision is
observed when the integrase is the only phage protein present. However,
100% excision is observed when the phage protein Orf7 is provided as
well as the integrase. Thus, Orf7 is the TP901-1 excisionase, and it is
the first excisionase identified that is used during excisive
recombination catalyzed by an integrase belonging to the family of
extended resolvases. Orf7 is a basic protein of 64 amino acids, and the
corresponding gene (orf7) is the third gene in the early
lytic operon. This location of an excisionase gene of a temperate
bacteriophage has never been described before. The experiments are
based on in vivo excision of specifically designed excision vectors
carrying the TP901-1 attP site which are integrated into
attB on the chromosome of Lactococcus lactis. Excision of the vectors was investigated in the presence of different TP901-1 genes. In order to detect very low frequencies of excision, a
method for positive selection of loss of genetic material based upon
the upp gene (encoding uracil phosphoribosyltransferase) was designed, since upp mutants are resistant to
fluorouracil. By using this system, frequencies of excision on the
order of 10 During the establishment of
lysogeny, many temperate bacteriophages integrate their genomes site
specifically into the bacterial host chromosome by recombination
between the attachment sites attB and attP,
located on the bacterial and phage genomes, respectively. This leads to
the formation of the hybrid attachment sites attL and
attR at the junctions between the phage and bacterial
genomes. At a later stage, induction of the phage will lead to
recombination between these sites and excision of the integrated phage
genome. Both integration and excision require the phage-encoded
integrase, but for efficient excision an additional phage-encoded
protein, the excisionase, is required. The excisionase counteracts the integration process and should therefore be expressed only during excision. This coordination of the expression of the integrase and
excisionase proteins is obtained by different mechanisms in different
phages. In Escherichia coli phage In bacteriophage P2 of E. coli, the two major early
promoters, pC and pE, are
divergently located (9, 16). The protein product of the
first gene downstream of pC is the phage
repressor C, and the first gene downstream of pE
encodes the Cox protein, which both is the phage excisionase and is
involved in regulation of expression from pC and
pE (26, 32). Cox is thus a very important protein in the choice between the lytic and lysogenic responses of P2. Regulation of the activities of
pC and pE by C and Cox
results in only one of the promoters being active at a time. The gene
encoding the integrase of P2 is located further downstream of
pC, and thus expression of the integrase without concomitant expression of the excisionase is obtained when
pC is active and pE is
repressed. However, for excision of the prophage genome during
induction of the prophage, both the integrase and Cox are required. It
has been suggested that in this case pE is active, leading to expression of Cox, while at the same time
transcription of the integrase gene is initiated from a minor promoter
upstream of the integrase gene (33).
The lactococcal temperate bacteriophage TP901-1 is the subject of
investigation in the present study. Like that of P2, the TP901-1 genome
contains two major early promoters, termed pL
(for lytic) and pR (for repression)
(20). By analogy to P2, pL
corresponds to pE. The first gene downstream of
pL, orf5, encodes a protein which has
been demonstrated to be involved in regulation of the activities of
pR and pL (20)
and thus bears some resemblance to Cox of P2. Similarly,
pR corresponds to pC,
since the protein encoded by the first gene downstream of
pR is Orf4, which is required for repression of
both pL and pR during
lysogeny (20).
The gene encoding the TP901-1 integrase, Orf1, is located further
downstream of pR, followed by the
attP site. Most of the integrases of the temperate
bacteriophages infecting lactic acid bacteria which have been
identified are related to the integrase of the E. coli
bacteriophage This is the first report on the protein requirements for excisive
recombination for a phage encoding an integrase belonging to this
protein family. It is also the first identification of the excisionase
gene of a temperate bacteriophage of lactic acid bacteria. The
investigations have been performed by a new method for in vivo
determination of the frequency of excision, based on excision vectors
containing two marker genes, enabling selection both for the presence
(erm) and the absence (upp) of the vector. By
this method we have shown that the excisionase of TP901-1 is Orf7,
encoded by the third gene in the early lytic operon. TP901-1 is the
first temperate bacteriophage described in which the gene encoding the
excisionase is located at this position on the phage genome.
Bacterial strains and culture conditions.
Lactococcal
strains were grown without stirring at 30°C in M17 broth
(29) supplemented with 0.5% (wt/vol) glucose (GM17). Selection for 5-fluorouracil (FU)-resistant cells was performed in GSA
medium, which was prepared by supplementing the defined medium SA
(13) with 1% (wt/vol) glucose. FU was added to a final concentration of 10 µg/ml, erythromycin (ERM) was added to 2 µg/ml, and chloramphenicol (CAM) was added to 5 µg/ml. E. coli
cells were propagated at 37°C with stirring in Luria-Bertani broth
(24), and ERM was added to a final concentration of 150 µg/ml, CAM was added to 25 µg/ml, and ampicillin was added to 100 µg/ml. To prepare plates, all media were solidified by adding 1.5%
Bacto Agar.
Construction of plasmids.
The plasmids used in this study
are listed in Table 1. The upp
gene was amplified from pJM300 (22) by using primers pupp2-1 and pupp2-2 (Table 2) and cloned in
pGEM-3Zf(+), giving rise to pAB204. The upp gene was not
sequenced, but it encodes a functional product. Plasmid pAB211 contains
the upp gene of pAB204 cloned as an
XhoI-HindIII fragment in pIC-19R
(21). By inserting upp from pAB211 on a
BglII-PstI fragment at the
BglII-PstI sites in pTRKL2 (25),
pAB227 was constructed. Subsequently, excision vector pAB112 was
obtained by inserting a 1.1-kb SmaI fragment with the
upp gene of pAB227 in SmaI in pBF12, which is an
E. coli vector containing a 333-bp TP901-1 attP
fragment and the erm cassette (3). Plasmid
pAB174b was obtained by introducing the upp gene on an
XbaI-ApaI fragment in pLB44 (6). In
this case the upp gene was amplified by PCR with pJM300 as
the template and primers pupp1-1 and pupp1-2. Again, the PCR fragment
was not sequenced, but the upp gene encodes a functional
product. Excision vector pAB174a was obtained from pAB174b by digesting
with BamHI, inactivating the enzyme and religating, and
subsequently identifying a plasmid in which erm was
inverted relative to pAB174b.
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Novel Organization of Genes Involved in Prophage Excision
Identified in the Temperate Lactococcal Bacteriophage TP901-1
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
5 per cell could easily be measured. The
described selection principle may be of general use for many organisms
and also for types of deletion events other than excision.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
, the gene encoding the
excisionase, xis, is located upstream of and partially
overlapping the int gene, which encodes the integrase. In
the situation where only the integrase is required, transcription of
int is initiated at a promoter located within
xis, and when both the integrase and excisionase are
required, transcription is initiated from a promoter upstream of
xis (7).
(for example, see references 2, 15,
30 and 31), suggesting that the mechanism
of recombination is similar to that of the integrase. In contrast,
the integrase of temperate lactococcal bacteriophage TP901-1 belongs to
a family of recombinases which has been termed the extended resolvases (6). The N-terminal parts of the proteins of this family
show homology to the catalytic domains of the resolvases and invertases of site-specific recombinases, suggesting that the proteins perform recombination by a similar mechanism. However, instead of the short
DNA-binding domain present in the C-terminal part of the resolvases and
invertases, the extended resolvases contain an extension of about 300 amino acids or more.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Bacterial strains and plasmids used in this study
TABLE 2.
Oligonucleotides used in this study
DNA preparation.
Plasmid DNA was isolated from lactococcal
and E. coli cells by the alkaline lysis technique
(27). Lactococcal cells were treated with lysozyme at a
final concentration of 20 mg/ml for 20 min at 37°C, with shaking to
promote lysis, before the addition of NaOH. When required, the plasmid
DNA was further purified by applying the DNA on Qiagen columns as
recommended by the supplier (Qiagen Ltd., Hilden, Germany). Chromosomal
DNA from lactococcal cells was prepared as described for E. coli (27), except that the cells were frozen for 30 min
at 
80°C after harvesting, thawed to room temperature, and treated
with lysozyme at a final concentration of 20 mg/ml for 30 min at 37°C
to promote lysis.
Recombinant DNA techniques. DNA manipulations were performed by standard techniques (27). Restriction nuclease enzymes, T4 DNA ligase, and corresponding buffers were supplied by Pharmacia Biotech or New England Biolabs; all enzymes were used as recommended by the supplier. The Ampli taq DNA polymerase was used for PCR amplification of DNA fragments, with reaction conditions as recommended by the supplier (Perkin-Elmer Cetus). DNA sequences were determined as described previously (28), with modifications according to the instructions with the Thermo Sequenase Radiolabeled terminator cycle sequencing kit (Amersham Life Science). Oligonucleotides were supplied by T-A-G-Copenhagen, Copenhagen, Denmark, or by Pharmacia Biotech, Allerød, Denmark.
Transformation of E. coli and Lactococcus lactis. Plasmid DNA was introduced into E. coli cells by making the cells competent with CaCl2 and transforming as described previously (27). Lactococcal cells were made electrocompetent and transformed by electroporation as described previously (11).
Integration of excision vectors into attB of JM342. To obtain strain AB112, L. lactis subsp. cremoris JM342 (23) was transformed with 0.05 µg of pAB112 DNA and 0.05 µg of pLB81 DNA and plated on plates containing ERM. To ensure that pLB81 had been lost, leading to loss of the cat gene, the cells were tested on CAM plates. To obtain strain AB174a, JM342 was transformed with 0.1 µg of pAB174a DNA and plated on plates containing ERM. For both excision vectors, site-specific integration was verified by PCR with chromosomal DNA, demonstrating that the attB site had been lost and that attL and attR sites had been created (data not shown). The primers used for amplification of attB were pattB-R1 and pattBR-L1, the primers for attL were pattB-R1 and pattP-L1, and the primers for attR were pattBR-L1 and PB2 (Table 2).
Determination of the frequency of excision. To determine the effect of the open reading frames of a protein donor plasmid on the frequency of excision of an excision vector, the protein donor was introduced in the L. lactis subsp. cremoris JM342 strains containing the excision vectors integrated on the chromosome (strains AB112 and AB174a). After transformation, the cells were plated on plates containing CAM, to select for the incoming protein donor plasmid, but not ERM, allowing loss of the excision vector. Approximately 2,000 transformants were plated for each experiment. After growth at 30°C overnight, the colonies were pooled by being washed off the plates with 1 ml of 0.9% NaCl and were subsequently diluted in 0.9% NaCl. Appropriate dilutions were plated on GSA plates containing either only CAM (to determine the number of cells) or both FU and CAM (to determine the number of cells which had lost the excision vector and thus had become resistant to FU). When erm was used as the marker gene, FU was replaced by ERM in the GSA plates. Each experiment was repeated independently at least three times.
To investigate whether excision did take place, chromosomal DNA was prepared from the relevant FUr strains and used as the template in PCRs. The primers used were pattBL and BI-POB1inv, which amplify the attB region (Table 2).| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Excision vectors. To investigate the effect of selected TP901-1-encoded proteins on excision, specific excision vectors which enable accurate determinations of low frequencies of excision were designed. These vectors contain an E. coli origin of replication, the erm gene, and a region of the TP901-1 genome, including at least attP (Fig. 1). In the presence of the TP901-1 integrase, site-specific integration of these vectors into the attB site on the host chromosome can be obtained, as described for other attP-containing vectors (3).
|
upp tdk derivative of L. lactis subsp.
cremoris MG1363 (8). The gene product of
tdk catalyzes a step in an alternative pathway for the
conversion of uracil to dUMP, and JM342 is therefore resistant to 10 µg of FU per ml. Integration of an excision vector containing
upp in JM342 leads to sensitivity to FU and resistance to
ERM. A cell having lost the integrated vector can therefore be selected
for during the excision experiments by plating on GSA plates containing 10 µg of FU per ml; this can be further verified by testing for ERM sensitivity.
The integrase of TP901-1 is required for excision. The simplest excision vector (pAB112) used in this study carries a 333-bp TP901-1 fragment with attP (excision system I) (Fig. 2). This excision vector is integrated site specifically into the attB site of the recipient chromosome by donation of the integrase in trans, using cotransformation with an E. coli vector containing the orf1 gene (pLB81). Even though the orf1-containing plasmid is lost during growth of the recipient, enough integrase is produced to allow for integration of the excision vector, and the resulting strain is termed AB112.
|
6/CFU) thus
corresponds to the spontaneous mutation rate of the upp
gene. In the presence of pAJ43, the frequency of occurrence of
FUr cells increased 500-fold, showing that excision is
stimulated by the presence of orf1 to orf6 (Fig.
2). To investigate whether the integrase is needed for excision, an
amber stop codon was introduced into orf1. This mutation
completely abolished excision (pAJ41 in Fig. 2), showing that the
integrase is required for excision of TP901-1.
In some temperate bacteriophages the excisionase gene is located
immediately upstream of the integrase gene. Therefore, TP901-1 orf2 or orf3 could be expected to encode the
excisionase. However, when nonsense mutations were introduced into
these genes separately (data not shown) or when both genes were
inactivated by a deletion (pAB35), no reduction in the frequency of
excision was observed. Thus, neither Orf2 nor Orf3 is involved in
excision of TP901-1.
Plasmid pAB235 contains the same deletion in orf2 and
orf3 as pAB35, but in addition, a fragment upstream of
orf3, including orf4, pR,
pL, orf5, and orf6, is
deleted. When this plasmid was introduced in AB112, the frequency of
occurrence of FUr cells was slightly higher than the
upp mutation rate (Fig. 2). Some of the FUr
colonies obtained were found to be Erms, confirming that
excision did take place at a low frequency. This indicates that the
integrase of TP901-1 can catalyze a low but significant level of
excision without other phage-encoded proteins present and that even
though the pR promoter is not present upstream
of orf1 in pAB235, sufficient amounts of the integrase are
present to ensure a low frequency of excision.
Identification of the excisionase.
Since the amount of the
TP901-1 integrase produced from the phage fragment in pAB235 is
sufficient to enable a low frequency of excision, we constructed a
second excision system (system II) (Fig.
3), in which the excision vector
(pAB174a) contains the same TP901-1 fragment as pAB235. With this
excision system, protein donor plasmids not containing orf1
can be used. pAB174a was integrated into attB in JM342, and
the strain was termed AB174a. Surprisingly, an excision frequency of
3 × 104 FUr cell/CFU was observed in
AB174a when the only phage protein present was Orf1, encoded by the
integrated excision vector (Fig. 3). This frequency is 125-fold greater
than the excision frequency obtained with pAB235 as protein donor in
system I. The reason for the much higher excision frequency obtained
with AB174a could be that in system I the integrase is donated in
trans, whereas in system II it is donated in cis.
Furthermore, the excision frequency obtained with pCI3340 as protein
donor in system II is 150-fold higher than the mutation rate of the
upp gene, and from this it is concluded that the TP901-1
integrase alone is sufficient to obtain a significant frequency of
excision.
|
1
Ermr cell/CFU was obtained, showing that excision takes
place in 88% of the cells.
Since orf4 and orf5 have previously been found
not to have any effect on excision, it was then investigated whether
the high frequency of excision with pAB241 was due to orf6
or orf7 by introducing a deletion into orf6,
resulting in pAB243. This plasmid also resulted in a frequency of
excision of 1 FU cell/CFU when upp was used as the marker
gene (Fig. 3). By using erm as marker, a frequency of
5.1 × 102 Ermr cell/CFU was obtained,
showing that excision takes place in 94.9% of the cells. Thus, the
results strongly indicate that the high level of excision obtained with
pAB241 is caused by Orf7. To establish whether the effect is solely due
to the presence of Orf7, a plasmid (pAB245) containing orf7
transcribed from the pL promoter was constructed. Introduction of pAB245 in AB174a also led to 1 FUr cell/CFU, and with erm as the marker it led
to 2.6 × 104 Ermr cell/CFU; thus,
excision takes place in 99.97% of the cells. To verify that the
increased excision frequency is due to the expression of
orf7, pAB244 was constructed. This plasmid is similar to
pAB245, except that no promoter region is present upstream of
orf7. The introduction of pAB244 in AB174a did not lead to an increase in excision, demonstrating that transcription of
orf7 is necessary for the observed increase. In conclusion,
Orf7 and the integrase are the only phage-encoded proteins required for efficient excision of the TP901-1 prophage, and Orf7 is thus the TP901-1 excisionase.
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
New method for determination of frequencies of excision.
In
order to be able to monitor genetic events occurring at low
frequencies, a positive selection procedure is required. The upp gene allows for positive selection for loss of genetic
material encompassing the gene, and a upp mutation confers
resistance to FU in many organisms. The general outline of the
procedure used in the present study is therefore expected to be
functional in many different organisms and can be used for monitoring
the loss of any specific part of the genetic material, provided that a functional upp gene has been inserted and that an organism
with a proper genetic background is used. As shown in the present
paper, in L. lactis, a tdk upp host strain is a
good choice, since it is resistant to high levels of FU, thus making
the selection for FU resistant colonies very clean. By using the
present experimental procedures, excision events occurring with
frequencies just above 2 × 106 per cell can be measured.
Phage TP901-1-encoded proteins involved in excision.
In all
cases where the protein requirements for excision of a prophage have
been examined, the phage-encoded integrase catalyzes excision as well
as integration. However, in these phages the integrase belongs to the
integrase family of recombinases. Since the TP901-1 integrase
belongs to the extended resolvases, the protein requirement for
excision of the TP901-1 prophage might be different. However, in the
work reported here it was demonstrated that the TP901-1 integrase is
required for excision as it is in other temperate bacteriophages.
can perform excisive
recombination in vivo at a frequency of 0.2 to 4.5% of the frequency
obtained with the excisionase present (1). Also, the integrase-type integrases of bacteriophages ø13 and ø42 of
Staphylococcus aureus can catalyze excision alone
(4). The frequency is dependent on the amount of integrase
present, with the maximal frequencies observed being 0.5 and 1.5% of
full excision, respectively.
However, maximal excision of a TP901-1 based excision vector is
obtained only when the phage-carried orf7 is present in the cell, downstream of a promoter. Thus, it is concluded that Orf7 is the
TP901-1 excisionase. Orf7 is a small basic protein (7.5 kDa, 64 amino
acids, pI 9.80). No significant amino acid homology between TP901-1
Orf7 and other known excisionases or between Orf7 and other proteins in
the database is observed. This is in accordance with the observation
that phage excisionases are often small, basic proteins which have
little sequence homology.
Novel location of a phage excisionase.
The excisionase of
TP901-1 is encoded by the third gene downstream of the
pL promoter (19). This position of an
excisionase gene is unique among temperate phages. In the other cases
where the position of this gene is known, it is positioned either in the vicinity of int (exemplified by ) or as the first
gene of the early lytic operon (exemplified by P2).
| |
ACKNOWLEDGMENTS |
|---|
We thank Annette Johansen for constructing the plasmids pAJ43, pAJ41, pAJ39, and pAJ37 and for providing pAJ95 prior to publication. We thank Peter L. Madsen for pPM92, pPM115, and pPM129. We also thank Todd R. Klaenhammer for the gift of pTRKL2 and Gerald Fitzgerald for pCI3340 and pCI372. We sincerely appreciate the expert technical assistance of Lotte Bredahl and Lise Sørensen.
This work was supported by the FØTEK program through The Center of Advanced Food Studies and by grants from the EC STARLAB program (BIO4-CT96-0402) and the Carlsberg Foundation.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Microbiology, Technical University of Denmark, DK-2800 Lyngby, Denmark. Phone: 45 45 25 24 96. Fax: 45 45 88 26 60. E-mail: imkh{at}pop.dtu.dk.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
| 1. | Abremski, K., and S. Gottesman. 1981. Site-specific recombination. Xis-independent excisive recombination of bacteriophage lambda. J. Mol. Biol. 153:67-78[Medline]. |
| 2. | Boyce, J. D., B. E. Davidson, and A. J. Hillier. 1995. Sequence analysis of the Lactococcus lactis temperate bacteriophage BK5-T and demonstration that the phage DNA has cohesive ends. Appl. Environ. Microbiol. 61:4089-4098[Abstract]. |
| 3. |
Brøndsted, L., and K. Hammer.
1999.
Use of integration elements encoded by the temperate lactococcal bacteriophage TP901-1 to obtain chromosomal single-copy transcriptional fusions in Lactococcus lactis.
Appl. Environ. Microbiol.
65:752-758 |
| 4. | Carroll, D., M. A. Kehoe, D. Cavanagh, and D. C. Coleman. 1995. Novel organization of the site-specific integration and excision recombination functions of the Staphylococcus aureus serotype F virulence-converting phages ø13 and ø42. Mol. Microbiol. 16:877-893[Medline]. |
| 5. |
Christiansen, B.,
M. G. Johnsen,
E. Stenby,
F. K. Vogensen, and K. Hammer.
1994.
Characterization of the lactococcal temperate phage TP901-1 and its site-specific integration.
J. Bacteriol.
176:1069-1076 |
| 6. |
Christiansen, B.,
L. Brøndsted,
F. K. Vogensen, and K. Hammer.
1996.
A resolvase-like protein is required for the site-specific integration of the temperate lactococcal bacteriophage TP901-1.
J. Bacteriol.
178:5164-5173 |
| 7. | Echols, H., and G. Guarneros. 1983. Control of integration and excision, p. 75-92. In R. W. Hendrix, J. W. Roberts, F. W. Stahl, and R. A. Weisberg (ed.), Lambda II. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y |
| 8. |
Gasson, M. J.
1983.
Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing.
J. Bacteriol.
154:1-9 |
| 9. | Haggård-Ljungquist, E., K. Kockum, and L. E. Bertani. 1987. DNA sequences of bacteriophage P2 early genes cox and B and their regulatory sites. Mol. Gen. Genet. 208:52-56[Medline]. |
| 10. |
Hayes, F.,
C. Daly, and G. F. Fitzgerald.
1990.
Identification of the minimal replicon of Lactococcus lactis subsp. lactis UC317 plasmid pCI305.
Appl. Environ. Microbiol.
56:202-209 |
| 11. |
Holo, H., and I. F. Nes.
1989.
High-frequency transformation, by electroporation, of Lactococcus lactis subsp. cremoris grown with glycine in osmotically stabilized media.
Appl. Environ. Microbiol.
55:3119-3123 |
| 12. | Israelsen, H., S. M. Madsen, A. Vrang, E. B. Hansen, and E. Johansen. 1995. Cloning and partial characterization of regulated promoters from Lactococcus lactis Tn917-lacZ integrants with the new promoter probe vector, pAK80. Appl. Environ. Microbiol. 61:2540-2547[Abstract]. |
| 13. |
Jensen, P. R., and K. Hammer.
1993.
Minimal requirements for exponential growth of Lactococcus lactis.
Appl. Environ. Microbiol.
59:4363-4366 |
| 14. | Johansen, A. H. Unpublished data. |
| 15. |
Lillehaug, D., and N.-K. Birkeland.
1993.
Characterization of genetic elements required for site-specific integration of the temperate lactococcal bacteriophage øLC3 and construction of integration-negative øLC3 mutants.
J. Bacteriol.
175:1745-1755 |
| 16. |
Ljungquist, E.,
K. Kockum, and L. E. Bertani.
1984.
DNA sequences of the repressor gene and operator region of bacteriophage P2.
Proc. Natl. Acad. Sci. USA
81:3988-3992 |
| 17. | Madsen, P. L. 1996. Transcription of the lactococcal temperate phage TP901-1. Ph.D. thesis. University of Copenhagen, Copenhagen, Denmark |
| 18. | Madsen, P. L. Unpublished data. |
| 19. |
Madsen, P. L., and K. Hammer.
1998.
Temporal transcription of the lactococcal temperate phage TP901-1 and DNA sequence of the early promoter region.
Microbiology
144:2203-2215 |
| 20. | Madsen, P. L., A. H. Johansen, K. Hammer, and L. Brøndsted. 1999. Genetic switch regulating activity of early promoters of the temperate lactococcal bacteriophage TP901-1 J. Bacteriol., in press. |
| 21. | Marsh, J. L., M. Erfle, and E. J. Wykes. 1984. The pIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation. Gene 32:481-485[Medline]. |
| 22. |
Martinussen, J., and K. Hammer.
1994.
Cloning and characterization of upp, a gene encoding uracil phosphoribosyltransferase from Lactococcus lactis.
J. Bacteriol.
176:6457-6463 |
| 23. |
Martinussen, J., and K. Hammer.
1995.
Powerful methods to establish chromosomal markers in Lactococcus lactis: an analysis of pyrimidine salvage pathway mutants obtained by positive selections.
Microbiology
141:1883-1890 |
| 24. | Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y |
| 25. | O'Sullivan, D. J., and T. R. Klaenhammer. 1993. High- and low-copy-number Lactococcus shuttle cloning vectors with features for clone screening. Gene 137:227-231[Medline]. |
| 26. | Saha, S., E. Haggård-Ljungquist, and K. Nordström. 1987. The cox protein of bacteriophage P2 inhibits the formation of the repressor protein and autoregulates the early operon. EMBO J. 6:3191-3199[Medline]. |
| 27. | Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y |
| 28. |
Sanger, F.,
S. Nicklen, and A. R. Coulson.
1977.
DNA sequencing with chain-terminating inhibitors.
Proc. Natl. Acad. Sci. USA
74:5463-5467 |
| 29. | Terzaghi, B. E., and W. E. Sandine. 1975. Improved medium for lactic streptococci and their bacteriophages. Appl. Microbiol. 29:807-813. |
| 30. |
van de Guchte, M.,
C. Daly,
G. F. Fitzgerald, and E. K. Arendt.
1994.
Identification of int and attP on the genome of lactococcal bacteriophage Tuc2009 and their use for site-specific plasmid integration in the chromosome of Tuc2009-resistant Lactococcus lactis MG1363.
Appl. Environ. Microbiol.
60:2324-2329 |
| 31. | van Sinderen, D., H. Karsens, J. Kok, P. Terpstra, M. H. J. Ruiters, G. Venema, and A. Nauta. 1996. Sequence analysis and molecular characterization of the temperate lactococcal bacteriophage rlt. Mol. Microbiol. 19:1343-1355[Medline]. |
| 32. |
Yu, A., and E. Haggård-Ljungquist.
1993.
The Cox protein is a modulator of directionality in bacteriophage P2 site-specific recombination.
J. Bacteriol.
175:7848-7855 |
| 33. |
Yu, A.,
V. Barreiro, and E. Haggård-Ljungquist.
1994.
Regulation of int gene expression in bacteriophage P2.
J. Virol.
68:4220-4226 |
Journal of Bacteriology, December 1999, p. 7291-7297, Vol. 181, No. 23
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Haaber, J., Rousseau, G. M., Hammer, K., Moineau, S.
(2009). Identification and Characterization of the Phage Gene sav, Involved in Sensitivity to the Lactococcal Abortive Infection Mechanism AbiV. Appl. Environ. Microbiol.
75: 2484-2494
[Abstract] [Full Text] -
Denou, E., Pridmore, R. D., Ventura, M., Pittet, A.-C., Zwahlen, M.-C., Berger, B., Barretto, C., Panoff, J.-M., Brussow, H.
(2008). The Role of Prophage for Genome Diversification within a Clonal Lineage of Lactobacillus johnsonii: Characterization of the Defective Prophage LJ771. J. Bacteriol.
190: 5806-5813
[Abstract] [Full Text] -
Solem, C., Defoor, E., Jensen, P. R., Martinussen, J.
(2008). Plasmid pCS1966, a New Selection/Counterselection Tool for Lactic Acid Bacterium Strain Construction Based on the oroP Gene, Encoding an Orotate Transporter from Lactococcus lactis. Appl. Environ. Microbiol.
74: 4772-4775
[Abstract] [Full Text] -
Ghosh, P., Bibb, L. A., Hatfull, G. F.
(2008). Two-step site selection for serine-integrase-mediated excision: DNA-directed integrase conformation and central dinucleotide proofreading. Proc. Natl. Acad. Sci. USA
105: 3238-3243
[Abstract] [Full Text] -
Defoor, E., Kryger, M.-B., Martinussen, J.
(2007). The orotate transporter encoded by oroP from Lactococcus lactis is required for orotate utilization and has utility as a food-grade selectable marker. Microbiology
153: 3645-3659
[Abstract] [Full Text] -
Vegge, C. S., Brondsted, L., Neve, H., Mc Grath, S., van Sinderen, D., Vogensen, F. K.
(2005). Structural Characterization and Assembly of the Distal Tail Structure of the Temperate Lactococcal Bacteriophage TP901-1. J. Bacteriol.
187: 4187-4197
[Abstract] [Full Text] -
Lucet, I. S., Tynan, F. E., Adams, V., Rossjohn, J., Lyras, D., Rood, J. I.
(2005). Identification of the Structural and Functional Domains of the Large Serine Recombinase TnpX from Clostridium perfringens. J. Biol. Chem.
280: 2503-2511
[Abstract] [Full Text] -
Smith, M. C. A., Till, R., Brady, K., Soultanas, P., Thorpe, H., Smith, M. C. M.
(2004). Synapsis and DNA cleavage in {phi}C31 integrase-mediated site-specific recombination. Nucleic Acids Res
32: 2607-2617
[Abstract] [Full Text] -
Canchaya, C., Proux, C., Fournous, G., Bruttin, A., Brussow, H.
(2003). Prophage Genomics. Microbiol. Mol. Biol. Rev.
67: 238-276
[Abstract] [Full Text] -
Proux, C., van Sinderen, D., Suarez, J., Garcia, P., Ladero, V., Fitzgerald, G. F., Desiere, F., Brussow, H.
(2002). The Dilemma of Phage Taxonomy Illustrated by Comparative Genomics of Sfi21-Like Siphoviridae in Lactic Acid Bacteria. J. Bacteriol.
184: 6026-6036
[Abstract] [Full Text] -
Combes, P., Till, R., Bee, S., Smith, M. C. M.
(2002). The Streptomyces Genome Contains Multiple Pseudo-attB Sites for the {phi}C31-Encoded Site-Specific Recombination System. J. Bacteriol.
184: 5746-5752
[Abstract] [Full Text] -
Stoll, S. M., Ginsburg, D. S., Calos, M. P.
(2002). Phage TP901-1 Site-Specific Integrase Functions in Human Cells. J. Bacteriol.
184: 3657-3663
[Abstract] [Full Text] -
Crutz-Le Coq, A.-M., Cesselin, B., Commissaire, J., Anba, J.
(2002). Sequence analysis of the lactococcal bacteriophage bIL170: insights into structural proteins and HNH endonucleases in dairy phages. Microbiology
148: 985-1001
[Abstract] [Full Text] -
Breuner, A., Hammer, K.
(2001). Resolvase-like recombination performed by the TP901-1 integrase. Microbiology
147: 2051-2063
[Abstract] [Full Text] -
Lewis, J. A., Hatfull, G. F.
(2001). Control of directionality in integrase-mediated recombination: examination of recombination directionality factors (RDFs) including Xis and Cox proteins. Nucleic Acids Res
29: 2205-2216
[Abstract] [Full Text] -
Madsen, P. L., Johansen, A. H., Hammer, K., Brøndsted, L.
(1999). The Genetic Switch Regulating Activity of Early Promoters of the Temperate Lactococcal Bacteriophage TP901-1. J. Bacteriol.
181: 7430-7438
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»