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Applied and Environmental Microbiology, February 1999, p. 752-758, Vol. 65, No. 2
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Use of the Integration Elements Encoded by the Temperate
Lactococcal Bacteriophage TP901-1 To Obtain Chromosomal Single-Copy
Transcriptional Fusions in Lactococcus lactis
Lone
Brøndsted* and
Karin
Hammer
Department of Microbiology, Technical
University of Denmark, DK-2800 Lyngby, Denmark
Received 15 July 1998/Accepted 23 November 1998
 |
ABSTRACT |
Previously we showed that only one phage-expressed protein (Orf1),
a 425-bp region upstream of the orf1 gene (presumably
encoding a promoter), and the attP region are necessary and
also sufficient for integration of the bacteriophage TP901-1 genome
into the chromosome of Lactococcus lactis subsp.
cremoris (B. Christiansen, L. Brøndsted, F. K. Vogensen, and K. Hammer, J. Bacteriol. 178:5164-5173, 1996). In
this work, a further analysis of the phage-encoded elements involved in
integration was performed. Here we demonstrate that even when the
orf1 gene is separated from the attP region,
the Orf1 protein is able to promote site-specific integration of an attP-carrying plasmid into the attB site on the
L. lactis subsp. cremoris chromosome.
Furthermore, the first detailed deletion analysis of an
attP region of a phage infecting lactic acid bacteria was
carried out. We show that a fragment containing 56 bp of the attP region, including the core, is sufficient for the
site-specific integration of a nonreplicating plasmid into the
chromosome of L. lactis subsp. cremoris
when the orf1 gene is donated in trans. The
functional 56-bp attP region of TP901-1 is substantially
smaller than minimal attP regions identified for other
phages. Based on the deletion analysis, several repeats located within
the attP region seem to be necessary for site-specific
integration of the temperate bacteriophage TP901-1. By use of the
integrative elements (attP and orf1) expressed
by the temperate lactococcal bacteriophage TP901-1, a system for
obtaining stable chromosomal single-copy transcriptional fusions in
L. lactis was constructed. Two promoter-reporter integration vectors containing the reporter gene gusA or
lacLM, encoding 
-glucuronidase or -galactosidase,
respectively, were constructed. Immediately upstream of both genes are
found translational stop codons in all three reading frames as well as
multiple restriction enzyme sites suitable for cloning of the promoter
of interest. By transformation of L. lactis subsp.
cremoris MG1363 containing the integrase gene on a
replicating plasmid, the promoter-reporter integration vectors
integrated with a high frequency site specifically into the chromosomal
attachment site attB used by bacteriophage TP901-1.
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INTRODUCTION |
TP901-1 is a temperate phage for
which Lactococcus lactis subsp. cremoris 3107 is
the host. During infection, the phage genome can integrate site
specifically into the bacterial chromosome by recombination between
attachment sites attB and attP located on the
bacterial and the phage genomes, respectively. This process leads to
the formation of the hybrid attachment sites attL and attR at the junctions between the phage and the bacterial
genomes. In all attachment sites (attB, attP,
attR, and attL), the 5-bp core region in which
recombination occurs is present. In addition, a 7-bp identical region
is present in all four attachment sites, separated from the core region
by a 1-bp mismatch (8).
Upstream of the attP region is located an open reading frame
(orf1) which encodes a 485-amino-acid protein (Orf1). Orf1
is a member of a new family of site-specific recombinases which are more than twice the size of the resolvases but which show significant similarity to the resolvases in the N terminus of about 150 amino acids
(9). The family of extended resolvases contains three more
integrases encoded by bacteriophages: the Sre protein of phage R4 of
Streptomyces parvulus, Orf613 of phage 
C31 of
Streptomyces fradia, and part of an open reading frame of
Bacillus cereus bacteriophage TP21 (17, 21, 24).
Also included are the site-specific recombinases from Bacillus
subtilis and several Anabaena species involved in chromosomal inversion and deletion events occurring during
differentiation into spores or heterocysts as well as the resolvase
(TnpX) of the conjugative chloramphenicol resistance
transposon Tn4451 from Clostridium
perfringens (3, 7, 32). Identified integrases of other
temperate lactococcal bacteriophages (Tuc2009, r1-t, LC3, and BK5-T)
are all of the Int type, showing homology to the integrase of
Escherichia coli bacteriophage 
(5, 19, 34,
35). Orf1 is thus a unique type of integrase among temperate lactococcal bacteriophages.
The study of gene expression and gene regulation in lactic acid
bacteria has been carried out mainly by use of transcriptional fusions
located on replicating plasmids. In these studies, the variation in the
copy number of the plasmids under different physiological conditions
and in different mutants was not taken into account. By maintenance of
the transcriptional fusions in single copies on the chromosome, the
effects of plasmid copy number can be avoided. Several systems for
the integration of genes into the chromosomes of lactic acid
bacteria have been described, but none of these have been specifically
designed for the study of gene expression and regulation (2,
4, 20). Only Sanders et al. (30) described a
method for the construction of chromosomal lacZ
transcriptional fusions by homologous recombination.
Previously we showed that only one phage-expressed protein (Orf1), a
425-bp region upstream of the orf1 gene (presumably encoding a promoter), and the attP region are necessary and
also sufficient for integration of the phage TP901-1 genome into
the chromosome of L. lactis subsp. cremoris
(9). In this work, we performed a detailed deletion analysis
of the attP region. Furthermore, we describe a method
for stable site-specific integration of transcriptional fusions
into the chromosome of L. lactis. The system is based on the phage-encoded elements (the attachment site attP and
the integrase gene orf1) necessary for integration of the
temperate lactococcal bacteriophage TP901-1 into the chromosome of
L. lactis.
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MATERIALS AND METHODS |
Cell growth and enzyme assay.
Lactococcus strains were
propagated at 30°C in M17 broth (Oxoid Limited, Basingstoke,
Hampshire, United Kingdom) containing 0.5% (wt/vol) glucose without
shaking (33). E. coli strains were grown with
agitation at 37°C in Luria-Bertani broth (Difco Laboratories,
Detroit, Mich.) (29). Bacto Agar (Difco) was used at 1.5%
(wt/vol) in solid media. For determination of -galactosidase activity, cells were permeabilized with sodium dodecyl sulfate (0.1%)
and chloroform. Cell debris was removed by high-speed centrifugation. The assay was performed as described by Miller (25).
DNA technology.
Extraction of chromosomal DNA was performed
as described for E. coli (29), with the
modification that cells were treated with 20 µg of lysozyme per ml
for 2 h before lysis. Recombinant plasmid DNA from E. coli was isolated by the alkaline lysis technique, and preparative
portions were further purified on Qiagen (Hilden, Germany) columns as
recommended by the supplier. Restriction endonuclease enzymes, DNA
polymerase Klenow fragment, T4 DNA ligase, and buffer systems were
supplied by Pharmacia Biotech. All enzymes were used as recommended by
the supplier. The PCR was performed by use of a DNA thermal cycler
(Perkin-Elmer Cetus) with Amplitaq polymerase and buffer supplied from
Perkin-Elmer Cetus.
Plasmid DNA for sequencing was prepared from E. coli DH5
.
The DNA sequences were determined by the method of Sanger and coworkers (31) with a Sequenase version 2.0 DNA sequencing kit (U.S.
Biochemical Corp., Cleveland, Ohio).
Construction of plasmids.
The plasmids used in this study
are listed in Table 1. By use of TP901-1
DNA as a template and primers PB2 and PB3, a 333-bp attP PCR
fragment was produced and cloned into the pMOSBlue vector (Amersham Life Science). Subsequently, the erm gene (1.1 kb)
from pUC7,erm was cloned into the EcoRI site,
resulting in plasmid pBF12 containing a 333-bp attP region
(attP1-333) (Fig. 1). Plasmids pBF17a and
pBF17b were constructed as follows. A purified 237-bp HincII
fragment from pBF12 was cloned into a purified 3.9-kb pBF12 fragment
digested with SmaI and XbaI, and Klenow polymerase was used for filling in. The orientation of the 207-bp attP region was the same in plasmid pBF17a
(attP127-333) (Fig.
1) as in pBF12, whereas in pBF17b
(attP127-333) (Fig. 1) the same attP
region was cloned in the opposite orientation. Plasmid pBF18
(attP173-333) (Fig. 1) was constructed by religation of a 4.1-kb purified EcoRV- and
SmaI-digested pBF12 fragment; pBF18 therefore contains 161 bp of the attP region. Plasmid pBF20 was constructed by
cloning a 102-bp purified AseI- and KpnI-digested
fragment from pBF12 into a digested (NdeI and KpnI) and purified 3.9-kb fragment from pBF12; plasmid pBF20
therefore contains 102 bp of the attP region
(attP173-274) (Fig. 1). Plasmids pBF26, pBF27,
and pBF30 contain 89 bp (attP186-274), 59 bp
(attP216-274), and 56 bp
(attP186-241) of the attP region,
respectively (Fig. 1), and were constructed by cloning of PCR fragments
produced with pBF12 as the template. For construction of plasmids pBF26
and pBF27, PCR fragments were produced with primers PB1 and PB4 or
primers T7 and PB5, respectively. These PCR fragments were digested
with AseI, purified, and cloned into a purified 3.9-kb
SmaI- and NdeI-digested pBF12 fragment, resulting in plasmids pBF26 and pBF27. For construction of plasmid pBF30, a PCR
fragment was produced with primers PB6 and PB4. This PCR fragment was
digested with SphI and EcoRV, purified, and
cloned into a purified 3.9-kb SphI- and
SmaI-digested fragment from pBF12. All attP
fragments, except those in pBF17b, were cloned in the same orientation
within the vector, and the presence of the respective attP
regions was confirmed by sequencing.
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FIG. 1.
Sequence of the attP region of the temperate
bacteriophage TP901-1. The bold sequence shows the coding region for
the integrase of TP901-1 (Orf1). The core region is boxed, and the
identical region is underlined. Direct and inverted repeats are
indicated with black arrows above the sequence. Repeats identified by
Christiansen et al. (8) are R1, R2, R3, R4, R5, and R6,
whereas the recently identified inverted repeat is P1. The numbers and
small arrows under the sequence indicate the borders of the different
attP fragments. The largest attP fragment
contains all bases shown, e.g., 1 to 333. Base 1 corresponds to base
2499 in the sequence deposited in GenBank.
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|
Plasmids containing the
orf1 gene without the
attP region of TP901-1 were constructed as follows. Plasmid
pAB201 contains
the
orf1 gene without the upstream sequence
and the
attP region.
Plasmid pLB61 was constructed by
ligation of a 3.4-kb
EcoRI-
ClaI
fragment of
pAB201 and a 1.6-kb
EcoRI-
ClaI fragment of pLB45.
A 1.9-kb
EcoRI-
SalI fragment from pLB61 was
cloned into shuttle
vector (both
E. coli and
L. lactis) pCI372 (
12), giving rise
to plasmid pLB65, and
into
L. lactis vector pGhost8 (
22), which
carries a temperature-sensitive origin of replication, giving
rise to
plasmid pLB95. Plasmids pLB65 and pLB95 both contain the
orf1 gene and a 425-bp region upstream of the
orf1 gene but not
the
attP core region usually
located downstream of the
orf1 gene.
The promoter-reporter integration vector pLB85 was constructed as
follows. A 1.8-kb purified
PstI-
HindIII
fragment from pNZ273
carrying the
gusA gene as well as stop
codons in all three reading
frames was cloned into
PstI- and
HindIII-digested pBF17a. The
promoter-reporter
integration vector pLB86 was constructed by
cloning a purified 4.0-kb
HindIII-
SalI fragment from pAK80 containing
the
lacL and
lacM genes (
15) into
plasmid pBF17b. Integration
vectors carrying promoter regions were
constructed as follows.
From pCP15 and pCP22 (
16), 4.1-kb
HindIII-
SalI fragments carrying
the
constitutive promoters and the reporter genes
lacL and
lacM were cloned into pBF17b, giving rise to pLB88 and
pLB87, respectively.
Plasmid pLB89 was constructed by cloning a 4.9-kb
HindIII-
SalI
fragment from pJM334
(
23) into
pBF17b.
Primers used in this study.
The primers used for PCR
amplification of fragments for cloning were as follows: PB1
(5'-CCTTCTATGCATGAGATAAC-3'), PB2
(5'-GCTGCTTAAAGCTAAGATT-3'), PB3
(5'-GCAAATTTCACAGATCGATA-3'), PB4
(5'-GGGGGCTCGAGTCCAACTCGCTTAATTGC-3'), PB5
(5'-GGGGGCTCGAGCGTTTATTTCAATTAAGGTAAC-3'), PB6
(5'-GGGGGGCATGCTTTAGTTACCTTAATTGAAAT-3'), and T7
(5'-TATACGACTCACTATAGGG-3').
The primers used for PCR amplification of
attB were as
follows: pattBL (5'-CTACTGCTGCTTCACCAG-3') and BI-POB1inv
(5'-GTATGCAGCGATGTCGTTACCC-3').
The primers used for PCR
amplification of
attL and
attR of integrated
pLB85 and derivatives thereof were as follows: pattBL and gusArev
(5'-GTCGAGTTTTTTGATTTCACGGG-3') (for
attL) and
BI-POB1inv and
1211 (5'-GTAAAACGACGGCCATG-3') (for
attR). The primers used for
PCR amplification of
attL and
attR of integrated pLB86 and derivatives
thereof were as follows: pattBL and 1211 (for
attL) and
BI-POB1inv
and PB5 (5'-CGTTTATTTCAATTAAGGTAAC-3') (for
attR).
Transformation and selection in E. coli and
L. lactis subsp. cremoris.
E. coli
DH5 [80lacZ
M15
(lacZYA-argF)U169 recA1 endA1 hsdR17 supE44 thi-1
gyrA96 relA1] (laboratory strain) was made competent with
CaCl2 and transformed as described by Sambrook et al.
(29). When the erm cassette was introduced,
selection was performed with 100 µg of ampicillin per ml and 150 µg
of erythromycin per ml.
L. lactis subsp.
cremoris MG1363
(
10) was transformed by electroporation by the method of
Holo and Nes (
13) with 0.03
to 0.5 µg of DNA per
electroporation. Transformants were selected
on plates containing 2 µg of erythromycin per ml. Integration
was analyzed as the presence
of
attL and
attR and the absence
of
attB in chromosomal DNA from representative transformants by
PCR as previously described (
8). The DNA concentration of
the
plasmid preparations was determined by comparing the digested
plasmid DNA with known amounts of
DNA digested with
HindIII
(Boehringer Mannheim
GmbH).
Nucleotide sequence accession number.
The attP
sequence reported in this study has been deposited in GenBank under
accession no. X85213.
| |
RESULTS |
Deletion analysis of the attP region of TP901-1.
In E. coli bacteriophage , several proteins involved in
the recombination process bind to repeats present within the
attP region. One of these proteins is the phage-expressed
integrase responsible for the actual crossing over of the DNA strands,
whereas other proteins present in the host are involved in changing the DNA conformation: IHF (integration host factor), FIS (factor for inversion stimulation), and XIS (excise) (for a review, see
reference 18). The attP region of the
temperate bacteriophage TP901-1 contains, in addition to the 5-bp
core region and a 7-bp region also present in the attachment sites
attB, attL, and attR, several direct
and inverted repeats surrounding and overlapping the core region (Fig.
1). These repeats could be binding sites for proteins involved in
recombination between the attP and attB sites
during integration of the TP901-1 phage genome into the bacterial
chromosome. By deletion analysis, the importance of the repeats located
in the attP region for the integration process was
investigated. Several of the repeats are located within orf1
(Fig. 1), encoding the integrase of TP901-1, which mediates
recombination between the attP and attB sites. In
order to make deletions in the attP region without
interfering with orf1, the attP region was
separated from the orf1 gene.
All plasmids which carry different parts of the
attP region
are derivatives of
E. coli vectors containing a selectable
marker
(
erm) functional in
Lactococcus but no
origin of replication functional
in gram-positive bacteria. The
integrase is donated in
trans by
plasmid pLB65, which
carries both the expressed
orf1 gene without
the
attP region and the
cat gene and is able to
replicate in
L. lactis subsp.
cremoris. The
ability of all
attP plasmids to integrate
into the
chromosomal
attB site was investigated by transformation
of
L. lactis subsp.
cremoris MG1363
containing pLB65 with the
respective
attP plasmids and
selection for erythromycin resistance.
As a control,
L. lactis subsp.
cremoris MG1363 containing the
vector
(pCI372) used for the construction of pLB65 was transformed.
Therefore,
the frequency of transformation of
L. lactis subsp.
cremoris MG1363 in the presence of either pLB65
(
orf1) or pCI372
(vector) could be calculated for all
attP-carrying plasmids (Table
2). In the presence of pLB65
(
orf1), the frequency of transformation
varied from 5 × 10
6 to 2 × 10
7 CFU/µg of DNA
for all
attP plasmids, except pBF27, which resulted
in a
frequency of transformation of 10
4 CFU/µg of DNA. In the
presence of pCI372 (vector), the frequency
of transformation varied
from 2 × 10
2 to 2 × 10
4 CFU/µg of
DNA for all
attP plasmids (Table
2).
The percentage of insertions due to site-specific integration was
calculated for all
attP plasmids. This value was found to
be
99.9% for all
attP plasmids, except pBF27, for which this
value
was 0%. This result suggests that in the case of pBF27, all
transformants
obtained in the presence of Orf1 were caused by the
presence of
the vector DNA, indicating that pBF27 was not able to
integrate
site specifically. It is possible that these transformants
arose
from homologous recombination between almost identical regions
located in both the vector (pCI372) and the vector part of pBF27.
The
same could also be true for transformants that arose from
the
transformation of
L. lactis subsp.
cremoris
containing pCI372
(vector) with any of the
attP plasmids. By
PCR performed on chromosomal
DNA extracted from chloramphenicol- and
erythromycin-resistant
transformants carrying either pLB65
(
orf1) or pCI372 (vector),
site-specific integration of the
attP plasmids was verified by
the presence of
attL and
attR as well as the absence of
attB (data
not shown). When
L. lactis subsp.
cremoris MG1363 carried only
the vector (pCI372), none of
the
attP plasmids was able to integrate
site specifically,
whereas in the presence of pLB65 (
orf1), site-specific
integration could be verified for all
attP plasmids, except
pBF27.
The results concerning the site-specific integration of the
attP plasmids are summarized in Fig.
2, which combines the structure
of the
plasmids containing various deletions of the
attP region
and
their ability to integrate site specifically into the chromosome
of
L. lactis subsp.
cremoris MG1363. Plasmid
pBF30 harbors the
smallest functional
attP fragment
(
attP186-241) (Fig.
1),
containing the
inverted R5 repeats and the overlapping R1 and
P1 repeats, as well as
the R4 and P1 repeats overlapping the core
region; in contrast, 14 repeats are found in the largest plasmid
(pBF12). When pBF26
(
attP186-274) (Fig.
1) and pBF27
(
attP216-274)
(Fig.
1) were compared, the
former was able to integrate, while
the latter was not. This result
strongly indicates that the R5
repeats and/or the overlapping R1 and P1
repeats are necessary
for site-specific integration.
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FIG. 2.
Deletion analysis of the attP region of the
temperate bacteriophage TP901-1. For each plasmid, the nucleotides of
the cloned attP fragment are indicated by numbers, which
correspond to the numbers in Fig. 1. The cloned attP
fragment is indicated by a black line, and the ability of each fragment
to promote site-specific integration of the plasmid is indicated (yes
or no). The large black arrow indicates the 3' end of the
orf1 gene, the black box indicates the core region, and
small arrows indicate direct and inverted repeats.
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Promoter-reporter vectors for site-specific integration.
Two
promoter-reporter integration vectors containing the reporter gene
gusA or lacLM, encoding -glucuronidase or
-galactosidase, respectively, were constructed. Immediately upstream
of both genes, translational stop codons in all three reading frames
are found. These genes have been used for the study of gene
expression and regulation on multicopy plasmids in L. lactis (15, 28). The integration vectors contain an
E. coli origin of replication, a selectable marker (the
erm gene) for Lactococcus, the bla
gene, and a 207-bp fragment carrying the attP region
(attP127-333) (Fig. 1) of the temperate
bacteriophage TP901-1. In addition, multiple cloning sites, which are
suitable for cloning of the promoter of interest, are located
immediately upstream of the reporter genes (Fig.
3A).
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FIG. 3.
(A) Structures of the promoter-reporter integration
vectors pLB85 and pLB86. The direction of transcription is indicated by
arrows. The black box indicates the 207-bp attP region of
TP901-1. (B) Results of site-specific integration of the
promoter-reporter vectors in the chromosome of L. lactis subsp. cremoris. The multiple cloning sites
(mcs) are indicated by horizontal lines, and the direction of
transcription is indicated by arrows. The black box indicates the
attP part of attL and attR of TP901-1,
and the thin black arrow indicates the orientation of transcription of
chromosomal genes located within the attB region
(8).
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The promoter-reporter integration vectors could integrate into
the
attB site of
L. lactis subsp.
cremoris MG1363 when the
integrase of TP901-1 was
present.
L. lactis subsp.
cremoris MG1363
containing the integrase gene on a replicating plasmid (pLB65)
was transformed with the promoter-reporter integration vectors
(pLB85
and pLB86). In addition,
L. lactis subsp.
cremoris MG1363
containing the integrase gene on a plasmid
carrying a temperature-sensitive
origin of replication (pLB95) was
transformed with the promoter-reporter
integration vectors (pLB85 and
pLB86). By selection for erythromycin-resistant
transformants,
approximately 5 × 10
6 CFU/µg of DNA was obtained in
the presence of the integrase gene
in pLB65, whereas in the presence of
pLB95 (temperature-sensitive
origin), approximately 5 × 10
4 CFU/µg of DNA was found. A strain carrying
pLB95 can be cured
of this plasmid by growth without selection at
37°C. Subsequently,
the loss of pLB95 can be confirmed, since the
strain becomes sensitive
to
tetracycline.
In the presence of the integrase of TP901-1, the promoter-reporter
integration vectors were integrated site specifically into
the
chromosomal attachment site
attB used by bacteriophage
TP901-1.
This finding was verified by the presence of
attL
and
attR as
well as the absence of
attB in a PCR
analysis performed on chromosomal
DNA extracted from eight
independent erythromycin-resistant transformants
(data not
shown). Furthermore, no PCR product was found with primers
annealing at each side of the
attP site, showing that only
one
copy of the vector was integrated site specifically into the
chromosome
(data not shown). When the promoter-reporter vectors were
integrated
into the
attB site of the chromosome,
transcription of the reporter
genes was divergent relative to the
transcription of the adjacent
chromosomal gene (Fig.
3B)
(
8).
To test the system, different promoter regions were cloned into the
promoter-reporter vector pLB86 and integrated site specifically
into
the
attB site on the chromosome of
L. lactis
subsp.
cremoris MG1363. The promoter activity of the
integrated fusions was measured
as
-galactosidase activity in
overnight cultures. The activity
of the chromosomal single-copy
promoter-reporter fusions was decreased
six- to ninefold compared with
that of the plasmid-borne promoter-reporter
fusions (Table
3).
To test the stability of the integrated fusions,
L. lactis subsp.
cremoris MG1363 containing plasmid pLB89
(the
upp promoter
cloned in front of the
lacLM
gene) integrated into the
attB site
on the chromosome was
grown without selection. To be able to screen
for the loss of the
integrated promoter-
lacLM transcriptional
fusion, cultures
were plated on plates containing
5-bromo-4-chloro-3-indolyl-
-
D-galactopyranoside
(X-Gal)
at selected times. After growth for more than 100 generations,
no
white colonies were found among the approximately 10,000 colonies
screened, showing that the integrated fusion was stably maintained
within the
attB site of
L. lactis subsp.
cremoris MG1363.
| |
DISCUSSION |
In this study, we performed a detailed deletion analysis of the
attP region of the temperate lactococcal bacteriophage
TP901-1. This deletion analysis is the first reported for phages
infecting lactic acid bacteria. In our analysis, the smallest
functional attP region found was a 56-bp fragment carrying
the core region and several repeats.
The 56-bp attP region contains the R5 inverted repeats, one
repeat each of R4 and R1, and the P1 inverted repeats. Deletion of the
R5 inverted repeats and the overlapping P1 and R1 repeats leads to a
nonfunctional attP region, suggesting that one or more of
these repeats are necessary for site-specific integration of the
temperate bacteriophage TP901-1. Furthermore, a second P1 repeat
located within the core region is expected to bind the integrase
of TP901-1 during recombination. Thus, the P1 repeats could be binding
sites for the integrase of TP901-1. Sequences showing homology to
the P1 repeats were also found close to and within the attB
core region on the L. lactis subsp.
cremoris chromosome (6).
The 56-bp attP region of TP901-1 is substantially smaller
than identified minimal attP regions reported for other
phages, such as bacteriophage (235 bp), bacteriophage P2 (220 bp),
and bacteriophage HP1 (418 bp) (11, 14, 26, 36). These three minimal functional attP regions all contain several sites
for the binding of different proteins involved in integration. These proteins are, in addition to the integrase which catalyzes the crossing
over of the DNA strands, DNA binding proteins which introduce bends in the DNA. In bacteriophage , the DNA-bending proteins, the
integrase, and the attP region form a complex, called the intasome, which makes recombination between the attP and
attB regions possible (for a review, see reference
18). The small size of a functional attP
region of TP901-1 suggests that a limited number of proteins are
involved in the integration process. Furthermore, the integrase of
TP901-1 does not show homology to the integrase of but belongs to a
new family of recombinases (the extended resolvases) which contains a
region showing homology to the catalytic site of resolvases and
invertases but which contains an extended C terminus (9).
These details suggest that recombination between the attP
and attB sites catalyzed by the TP901-1 integrase,
representing the new family of recombinases, requires the binding of
less auxiliary proteins than the integrase. However, as has
been found for the integrase, site-specific integration mediated by
the TP901-1 integrase can be performed when the integrase is
donated in trans.
Based on the phage-encoded elements (the attachment site
attP and the integrase gene orf1) necessary for
integration of the temperate bacteriophage TP901-1 into the chromosome
of L. lactis, we have developed a method for the
site-specific integration of transcriptional fusions into the
chromosome of L. lactis. A similar system based
on the integrative elements for bacteriophage for use in
E. coli has been developed by Atlung et al. (1).
Two promoter-reporter integration vectors containing the reporter gene
gusA or lacLM, encoding -glucuronidase or
-galactosidase, respectively, were constructed. The two vectors
integrated site specifically into the chromosomal attB site
used by TP901-1 in the presence of the TP901-1 integrase. The system is
suitable for the study of gene expression and regulation in
L. lactis, since the transcriptional fusion is stably
maintained in a single copy within the chromosome, thereby eliminating
the effects of variation of plasmid copy number. Furthermore, by
integration of promoter-reporter transcriptional fusions in the TP901-1
phage attachment site on the chromosome, the promoter fragments are not
located in the usual context. With this system, it is therefore possible to study the effect of deletions on promoter activity and
regulation; such study is not straightforward when the construction of
chromosomal transcriptional fusions is based on homologous recombination (30).
| |
ACKNOWLEDGMENTS |
We are grateful to Willem M. de Vos for providing plasmid
pNZ273 prior to publication. We thank Anne Breüner for providing plasmid pAB201 and for stimulating discussions. Bjarne Faurholm is
acknowledged for construction of the pBF plasmids, and we sincerely appreciate the expert technical assistance of Lotte Bredahl.
This work was supported by grants from the EC BIOTECH-G program
(BIO2-CT94-3055) and the Carlsberg Foundation.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department
of Microbiology, Technical University of Denmark, Building 301, DK-2800 Lyngby, Denmark. Phone: 45 45 25 25 28. Fax: 45 45 88 26 60. E-mail: imlob{at}pop.dtu.dk.
| |
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Applied and Environmental Microbiology, February 1999, p. 752-758, Vol. 65, No. 2
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Top
Abstract
Introduction
