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Appl Environ Microbiol, March 1998, p. 1147-1152, Vol. 64, No. 3
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Common Elements Regulating Gene Expression in
Temperate and Lytic Bacteriophages of Lactococcus
Species
Shirley A.
Walker,
Carol S.
Dombroski, and
Todd R.
Klaenhammer*
Department of Food Science, Southeast Dairy
Foods Research Center, North Carolina State University, Raleigh,
North Carolina 27695-7624
Received 15 September 1997/Accepted 29 December 1997
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ABSTRACT |
A phage-inducible middle promoter (P15A10) from the
lytic, lactococcal bacteriophage 
31, a member of the P335 species,
is located in an 888-base pair fragment near the right cohesive end. Sequence analysis revealed extensive homology (>95%) to the right cohesive ends of two temperate phages of the P335 species, r1t and
LC3. Sequencing upstream and downstream of P15A10 showed that the high degree of homology between 31 and r1t continued beyond the phage promoter. With the exception of one extra open reading
frame in 31, the sequences were highly homologous (95 to 98%)
between nucleotides 13448 and 16320 of the published r1t sequence.
By use of a 
-galactosidase (-Gal) gene under the control of a
smaller, more tightly regulated region within the P15A10 promoter, P566-888, it was established that mitomycin C induction of a lactococcal strain harboring the prophage r1t induced
the P566-888 promoter, as determined from an increase in
-Gal activity. Hybridization of nine other lactococcal strains with
32P-labeled P566-888 showed that the
Lactococcus lactis strains C10, ML8, and NCK203 harbored
sequences homologous to that of the phage-inducible promoter. Mitomycin
C induced the resident prophages in all these strains and concurrently
induced the P566-888 promoter, as determined from an
increase in -Gal activity. DNA restriction analysis revealed that
the prophages in C10, ML8, and NCK203 had identical restriction
patterns which were different from that of r1t. In addition, DNA
sequencing showed that the promoter elements in the three phages were
identical to each other and to P566-888 from the lytic
phage 31. These results point to a conserved mechanism in the
regulation of gene expression between the lytic phage 31 and at
least two temperate bacteriophages and provide further evidence for a
link in the evolution of certain temperate phages and lytic phages.
| |
TEXT |
Lactococcus lactis
is an industrially important member of the lactic acid bacteria.
It is used in the fermentation of many dairy products, including sour
cream, buttermilk, and various cheeses such as cheddar. Lytic
bacteriophages routinely disrupt these industrial milk fermentations.
Despite the substantial research conducted to protect L. lactis, the appearance of new lytic phages continues to plague the
dairy industries (16, 27). In previous years, it was
observed that lytic and temperate phages showed little significant
homology at the DNA level (13). Therefore, it was not
considered likely that temperate phages contributed to the development
of lytic phages for L. lactis (5, 15). However,
the fairly recent emergence of the problematic P335 species (1,
19), composed of both lytic and temperate bacteriophages, has at
least partly refuted this position. The lytic and temperate phages of
the P335 species exhibit some DNA homology, suggesting that some
temperate and lytic phages may have common ancestors (4, 5, 15,
17, 19, 25). In addition, recent evidence suggests that prophages
may be an important source of DNA that contributes to the appearance of
new lytic phages (7, 20).
The lactococcal bacteriophage 31 is a small-isometric,
cohesive-ended, lytic lactococcal bacteriophage of the P335 species (1) with a double-stranded DNA genome of 31.9 kb. Recently, the first phage-inducible middle promoter from a lytic,
lactococcal bacteriophage was isolated from 31
(22). The 888-base pair promoter fragment
(P15A10) was cloned upstream of the -galactosidase (-Gal) gene (lacZ.st) from Streptococcus
thermophilus (28). The promoter yielded a low level of
-Gal activity before phage infection and was induced three- to
fourfold upon infection with phage 31. P15A10 was
subcloned to obtain a smaller, more tightly regulated phage promoter,
encompassed within nucleotides 566 to 888 (P566-888
[32]). This promoter fragment yielded -Gal activity
only after phage infection of the lactococcal host, NCK203 (10).
The phage-inducible promoter (P15A10), located near the
right cohesive end of the lytic bacteriophage 31 (22),
showed extensive DNA sequence homology (>96%) to two temperate
bacteriophages of the P335 species, r1t (31) and LC3
(3). This high level of homology led to an interest in the
distribution of this promoter in other temperate bacteriophages. The
goals of the present study were twofold: (i) to determine whether
or not other prophages harbored by lactococcal strains carried this
promoter element, and (ii) to evaluate if these prophages could
induce the P566-888 promoter, as determined from
expression of -Gal from a
P566-888::lacZ.st fusion in pTRK477
(Fig. 1) (32).
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FIG. 1.
Representation of pTRK477. The vector encodes the
-Gal gene (lacZ.st) from S. thermophilus
(28) under control of the tightly regulated middle promoter
(P566-888) from the lytic bacteriophage 31.
P566-888 was cloned as a BamHI fragment
(32) in the promoter screening vector pTRK390
(22). ori, origin of replication; Erm, erythromycin
resistance.
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Activation of P566-888 by r1t.
Phages 31
and r1t show extensive homology near the right cohesive
(cos) ends (22, 31). Therefore, L. lactis R1, the lactococcal strain harboring r1t in
its chromosome, was initially examined for activation of
P566-888 concurrent with prophage induction. R1Cs/r1t
(prophage positive) and R1Cs, a prophage-cured derivative,
were available from previous experiments (14). Both strains
were transformed with pTRK477
(P566-888::lacZ.st [Fig. 1]) by
the procedure of Holo and Nes (12) as modified by
Walker and Klaenhammer (32). One transformed colony was
obtained for R1Cs/r1t. Small-scale plasmid isolation (21)
and restriction analysis confirmed the presence of pTRK477. No
transformants were obtained for the cured derivative, R1Cs, even upon
repeated transformation attempts with increasing concentrations of
pTRK477. The vector pTRK477 was also isolated from the R1Cs/r1t
transformant and electroporated to R1Cs. No transformants were
obtained, indicating that a restriction/modification barrier was not
responsible. The reason for the difficulty in transforming R1 is not
known.
The unsuccessful attempts to transform R1Cs forced us to create a
prophage-negative control strain from R1Cs/r1t containing
pTRK477.
To cure
r1t, R1Cs/r1t (pTRK477) was propagated in GM17
(
30) at 30°C to an optical density at 600 nm
(OD
600) of ~0.2.
Erythromycin (EM) (2 µg/ml) was added
to maintain pTRK477. Mitomycin
C (2 µg/ml) was added to induce
r1t, and induction was allowed
to proceed for 45 min. The cells were
diluted in sterile phosphate-buffered
saline buffer and plated onto
GM17 containing 2 µg of EM per ml
[GM17 (EM)] and 1.5% agar so
that individual colonies could be
isolated. Individual colonies were
then patched onto fresh GM17
(EM) plates containing 10 mM
CaCl
2 and spotted with 10 µl of a
r1t lysate. The
cured derivatives were identified by their sensitivity
to
r1t. The
cure rate was greater than 15% for the survivors.
Plasmid mini-preps
and restriction analysis confirmed the presence
of pTRK477 in the cured
derivatives. The plasmid isolated from
R1Cs (pTRK477) was then
transformed into the original host,
L. lactis NCK203.
-Gal assays were performed during a phage
31
infection (
18,
22) to ensure that pTRK477 had not suffered
mutations during
mitomycin C treatment. No change in the level
of
-Gal induced from
the P
566-888 promoter was observed
(data not shown).
To evaluate whether or not
r1t could activate
P
566-888, R1Cs/r1t (pTRK477) and R1Cs (pTRK477) were
propagated in GM17
(EM) at 30°C to an initial OD
600 of
~0.2. Mitomycin C was then
added to a concentration of 2 µg/ml, and
growth continued for
4 h, with OD
600 readings and
-Gal activity assays performed every
30 min. The results are shown
in Fig.
2. R1Cs/r1t (pTRK477) showed
evidence of lysis within 3 h (lysis was complete upon overnight
incubation), while the cured derivative did not (Fig.
2A).
-Gal
activity was detected in R1Cs/r1t (pTRK477) upon induction with
mitomycin C and continued to increase during the lytic cycle (Fig.
2B).
No
-Gal activity was observed in the cured derivative. Therefore,
induction of phage
r1t activated the P
566-888
promoter.
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FIG. 2.
Induction of P566-888 by phage r1t. (A)
Growth of R1Cs/r1t and R1Cs, the prophage-cured derivative, in the
presence of mitomycin C. The strains were grown to an initial
OD600 of 0.2 before the addition of 2.5 µg of mitomycin C
per ml. Time zero represents the point of addition of mitomycin C. (B)
-Gal activity expression from R1Cs/r1t (pTRK477) and R1Cs (pTRK477)
after induction of the resident prophage with mitomycin C. -Gal
activity was measured by the procedure of Miller (18) as
modified by O'Sullivan et al. (22). Activity is expressed
as units/OD600 of the cell culture (22). -Gal
results reported are the averages of two separate assays. In each
assay, time points were performed (or assayed) in duplicate.
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Different Lactococcus prophages activate
P566-888.
To determine if other prophages
were capable of activating P566-888, several
lactococcal strains (Table 1) were tested for the presence of DNA homologous to the fragment from positions 566 to 888 by using slot blot Southern hybridization. As a negative control, the original r1t-cured derivative R1Cs (14),
which could not be transformed, was included. The probe was the
32P-labeled fragment from positions 566 to 888 (P566-888). Results are shown in Fig.
3. The DNA from three L. lactis subsp. lactis strains, NCK203, ML8, and C10,
showed homology to P566-888. These results were confirmed
by transferring HindIII digests of each DNA sample to a
MagnaCharge membrane (MSI, Westborough, Mass.) and probing with the
32P-labeled fragment P566-888 by using
standard procedures (26). Identical HindIII
fragments showing homology to P566-888 were observed for
all three strains (data not shown). DNA sequencing with the
Thermosequenase kit (Amersham Life Sciences, Arlington Heights, Ill.)
was then used to determine if the homologous DNA identified in
each strain by hybridization with P566-888 was
identical to that isolated from 31. The regions of C10, ML8, and NCK203 showing homology to P566-888 were amplified
by PCR from the genomes of C10, ML8, and NCK203 with one primer
consisting of nucleotides 566 to 582 and a second primer complementary
to nucleotides 865 to 888 of the published sequence from 31
(22, 32). The PCR products were then cloned into the T
vector, pGEM-T easy (Promega, Madison, Wis.) and transformed to JM110
(33) by the procedure of Hanahan (9) as modified
by Dinsmore and Klaenhammer (6). Sequencing was
performed with the T7 promoter primer, and the first 200 nucleotides of
each region (corresponding to nucleotides 566 to 766 of
P566-888) could be easily read. The sequences of the
homologous regions in the three prophages were identical to the
corresponding region of the P566-888 sequence of phage
31. The sequenced regions showed identical putative transcriptional
start sites, 
10 consensus regions, and inverted or direct repeats
positioned in the 35 regions (22, 32).
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FIG. 3.
Slot blot analysis of genomic DNA from 10 lactococcal
strains. Total DNA was isolated from each strain by the procedure of
Hill et al. (11). Slot blot Southern hybridizations were
performed with the Zeta probe membrane (BioRad, Richmond, Calif.) and a
BioRad apparatus according to the manufacturer's protocol. The probe
(the fragment from positions 566 to 888) was 32P-labeled
with the Multiprime DNA labeling system (Amersham Life Sciences).
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To determine whether or not prophages were present which could
activate P
566-888, each of the three strains was
transformed
with pTRK477 as described above for R1Cs/r1t. The strains
containing
pTRK477 were propagated in GM17 (EM) to an OD
600
of ~0.2. Mitomycin
C was added to 10 µg/ml to attempt induction of
the resident prophage.
Since repeated attempts to cure these
strains were unsuccessful,
cultures to which no mitomycin C was added
were used as controls
in this experiment. OD
600-monitored
lysis and
-Gal activity assays
were used to monitor the subsequent
induction of P
566-888.
Lysis of all three strains
began 2 h after the addition of mitomycin
C and was virtually
complete within 4 h. In addition, although
no
-Gal activity was
detected in the uninduced cultures,
-Gal
activity was induced to
approximately the same level (400 to 500
-Gal units) for all three
strains when mitomycin C was present.
These data provided strong
evidence that P
566-888 was activated
by induction of
the resident prophages.
DNA restriction analysis was used to determine if the prophages
from the lactococcal strains NCK203, C10, and ML8 were similar
to each
other or to
r1t. Prophage DNA was isolated as described
by Raya et
al. (
24). Briefly, each strain was propagated in
GM17 at
30°C to an OD
600 of ~0.25 to 0.3. Mitomycin C was then
added to a level of 10 µg/ml for NCK203 and ML8 and to a level
of 7.5 µg/ml for C10 and R1 (r1t) to induce the resident prophages.
After lysis, the sample was centrifuged, filter sterilized through
a
0.45-µm-pore-size filter to remove cell debris, and treated
with
DNaseI and RNaseA (1 µg of each per ml) for 30 min at 37°C.
The phages were then precipitated with NaCl (3%) and PEG 8000
(10%)
overnight, centrifuged at 8,000 rpm for 20 min, and resuspended
in 2 ml
of sterile distilled water. Phage DNA was isolated by
successive
phenol-chloroform-isoamyl alcohol extractions and precipitation
with
ethanol. DNA restriction analysis with
EcoRI clearly showed
that the three additional prophages identified by DNA hybridization
were similar to each other (identical restriction patterns) but
not to
r1t (Fig.
4). The similarity between
the temperate phages
isolated from C10, ML8, and NCK203 was also
confirmed with
HindIII
and
EcoRV (data not
shown).
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FIG. 4.
EcoRI digest patterns of DNA isolated from
the temperate phages of L. lactis C10, ML8, and NCK203. Lane
1, 1-kb ladder (Gibco-BRL, Gaithersburg, Md.); lane 2, lytic
bacteriophage 31; lane 3, r1t; lane 4, temperate phage isolated
from NCK203; lane 5, temperate phage from ML8; lane 6, temperate phage
from C10.
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Sequencing upstream and downstream of the phage-inducible
promoter.
Noting the extent of sequence homology of the
phage-inducible promoter, P15A10, to the temperate
bacteriophage r1t (22, 31), we also performed sequencing
upstream and downstream of the 15A10 fragment on phage 31 to
determine if this high level of homology continued. Phage 31 genomic
DNA was isolated as described earlier (24). Initially,
sequencing was performed directly on the phage 31 genome with
the fmol Cycle Sequencing Kit (Promega, Madison, Wis.) and a
32P-end-labeled primer (Table
2) according to manufacturer's
instructions. Most recently, cycle sequencing was performed with the
Thermosequenase kit (Amersham). This method utilizes
33P-labeled dideoxynucleotides to terminate sequencing
reactions on the 3' end, resulting in cleaner sequencing runs. To
sequence across the cos site, the phage 31 genome was
annealed and ligated by standard protocols (26).
Approximately 2,300 base pairs were sequenced downstream of
P15A10, and 360 base pairs were sequenced upstream.
Homology searches with the BLAST algorithm (2) revealed that
the entire region, with the exception of one open reading frame (ORF),
continued to show extensive DNA homology to r1t (95 to 98%
identity; nucleotides 13448 to 16320 of the published r1t sequence
[31]). At a site adjacent to the right cohesive end
(cos), which is identical to the r1t cos site,
the sequences diverge. In this region, 31 contains an additional ORF
not found in r1t (ORF4, 246 amino acids) (Fig.
5). The function of this protein is not
known. At the end of ORF4, the sequences of 31 and r1t converge
again (Fig. 5). A representation of the homology between 31 and
r1t in this region is shown in Fig. 6.
The functions of only two of the depicted putative ORFs have been
published. ORF2 (31) is the transcriptional activator for the
P566-888 promoter (32). ORF27 from r1t
has been identified as a putative structural protein (31).
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FIG. 5.
Representation of the DNA sequence of ORF4. The
sequences in gray represent areas with extensive homology to r1t.
The homology extends both upstream and downstream of this depicted
sequence, as described in the text. The solid underline denotes the
cohesive site of r1t. This exact sequence is present on 31. The
beginning of ORF4 is denoted by arrows. Two possible start codons, each
with a potential Shine-Dalgarno sequence upstream (broken line), exist
for ORF4. The first start codon is actually in the region of homology
to r1t. On r1t, a one-nucleotide difference in the region results
in a stop codon after the first three amino acids (codon TAG, resulting
in MGA). The second start codon is after the region of homology
(denoted by ORF4alt). The end of ORF4 overlaps the beginning of ORF5,
which shares extensive homology with r1t's ORF27, a putative
structural protein (31).
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FIG. 6.
Putative ORFs identified near the right cohesive end of
phage 31. The overlapping arrows of ORFs 4, 5, and 6 indicate that
the stop and start codons of these ORFs overlap each other. The extent
of homology to putative ORFs of r1t is shown in parentheses below
each phage 31 ORF. The positions of the cos site and the
phage-inducible promoter, P566-888, relative to the
identified ORFs are indicated by vertical arrows. The sizes of the ORFs
are as follows: ORF1, incomplete (161 amino acids [aa] sequenced);
ORF2, 143 amino acids; ORF3, 110 amino acids; ORF4, 216 or 246 amino
acids (two possible start sites); ORF5, 409 amino acids; and ORF6, the
nucleotides encoding the first 12 amino acids have been sequenced. The
accession number for this sequence is AF022773.
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Conclusions.
Due to the extensive homology of the phage 31
middle promoter to several temperate phages, this study investigated
whether or not other lactococcal strains harbored sequences homologous to that of P566-888. Of the nine strains tested, three
lysogenic strains were found to have homologous sequences. Mitocycin C
induction of a residing prophage in these strains
concurrently activated the P566-888 promoter,
generating -Gal activity. These results clearly point to a
conserved mechanism in the regulation of gene expression between the
lytic phage 31 and at least two temperate bacteriophages (r1t and
the residing prophage in C10, ML8, and NCK203). Therefore, lytic
and temperate phages of the P335 species appear to share a common
ancestor, strengthening the evidence for recent proposals (7,
20) that resident prophage sequences contribute to the
evolution of new lytic phages in the dairy lactococci.
Nucleotide sequence accession number.
The nucleotide
sequence of the region of 31 depicted in Fig. 6 has been
submitted to the EMBL databases under accession number AF022773.
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ACKNOWLEDGMENTS |
This research was supported, in part, by USDA/NRIGCP project
number 97-35503-4368 and by Rhone Poulenc, Inc., Madison, Wis. S.W. was
supported by a USDA-GAANN Biotechnology Fellowship.
We thank John McCormick, Evelyn Durmaz, and David Mills for helpful
discussions and for critical review of the manuscript.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Food Science, Southeast Dairy Foods Research Center, Box 7624, North Carolina State University, Raleigh, NC 27695-7624. Phone: (919) 515-2971. Fax: (919) 515-7124. E-mail: Klaenhammer{at}ncsu.edu.
Paper no. FSR 97-34 of the Department of Food Science, North
Carolina State University, Raleigh.
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