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Journal of Bacteriology, December 2001, p. 6965-6970, Vol. 183, No. 23
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.23.6965-6970.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Analysis of Early Promoters of the
Bacillus Bacteriophage GA-1
José A.
Horcajadas,1
Wilfried J. J.
Meijer,1
Fernando
Rojo,2 and
Margarita
Salas1,*
Centro de Biología Molecular
"Severo Ochoa" (CSIC-UAM)1 and
Centro Nacional de Biotecnología
(CSIC),2 Universidad Autónoma,
Cantoblanco, 28049 Madrid, Spain
Received 21 June 2001/Accepted 19 September 2001
 |
ABSTRACT |
Bacteriophage GA-1, which infects Bacillus sp.
strain G1R, is evolutionarily related to phage 
29, which infects
Bacillus subtilis. We report the characterization of
several GA-1 promoters located at either end of its linear genome. Some
of them are unique for GA-1 and drive the expression of open reading
frames that have no counterparts in the genome of 29 or related
phages. These unique promoters are active at early infection times and
are repressed at late times. In vitro transcription reactions revealed
that the purified GA-1-encoded protein p6 represses the activity of these promoters, although the amount of p6 required to repress transcription was different for each promoter. The level of protein p6
produced in vivo increases rapidly during the first stage of the
infection cycle. The protein p6 concentration may serve to modulate the
expression of these early promoters as infection proceeds.
| |
TEXT |
A large variety of phages that
infect bacteria of the genus Bacillus have been
characterized. Particular attention has been given to the so-called
29 family of phages that infect different Bacillus
species. The genome of these lytic phages consists of a small linear
double-stranded DNA of about 20 kbp, with a terminal protein covalently
linked to the 5' ends that plays a key role in the initiation of phage
DNA replication. On the basis of serological properties, DNA physical
maps, peptide maps, and partial or complete DNA sequences (26,
36, 37), the 29 family of phages has been classified into
three groups. The first group includes phages 29, PZA, 15, and
BS32; the second one includes B103, Nf, and M2Y; and the third group
has phage GA-1 as its sole member. Among them, phage 29 has been
extensively characterized, being one of the best-studied bacteriophages
of gram-positive bacteria. Its mechanism of DNA replication and its
regulation of transcription have been reviewed previously (19,
28, 29). Within this family of phages, GA-1 is the one most
distantly related to 29; this has stimulated the study of its
mechanisms of DNA replication (6, 7, 8, 12) and
transcription regulation (11).
The DNA sequences of the complete genomes of phages 29
(34), PZA (23), B103 (25), and
GA-1 (19) have been determined. The GA-1 genome has a size
of 21,129 bp, which is larger than those of 29 (19,285 bp), PZA
(19,366 bp), and B103 (18,630 bp). In most aspects, the genomes of
phages 29, B103, and GA-1 are similarly organized. In 29 (group
I) and B103 (group II), the genes expressed soon after infection (early
genes) are clustered in two operons located at each end of the genome.
The late genes are located in a single operon that is positioned at the
central part of the genome. As shown schematically in Fig.
1, the late genes of GA-1 (genes 7 through 16) are also present in a single operon located in the central
part of the genome. As in 29 and B103, the late GA-1 operon is
flanked on its left side by an early operon that contains genes
necessary for DNA replication and for transcriptional regulation (genes
6 through 2). These genes are expressed from the early promoters A2b
and A2c (11). The right region of the GA-1 genome contains
open reading frames (ORFs) whose deduced protein sequences are
homologous to those of the 29 early genes 17 and 16.7, which are
involved in DNA replication (5, 17, 18). However, both
ends of the GA-1 genome contain a number of sequences and ORFs that
have no counterparts in 29 or in any of the other related phages
characterized (19). Therefore, the proteins that are
probably encoded by these ORFs are unique for GA-1. Several putative
promoters that could be responsible for the expression of these unique
ORFs were identified. In this work we characterized these promoters,
analyzing their expression patterns throughout the infection cycle. We
also analyzed the role of GA-1 protein p6 in the regulation of the
early promoters in vitro.
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FIG. 1.
Genetic and transcriptional map of phage GA-1 genome.
The terminal protein (TP) is shown attached to the 5' ends of the DNA.
Promoters are indicated; the start sites of promoters A1c, A1a, C1, and
C2 correspond to those determined in this work. Genes with known
function, or for which counterparts are present in phage 29, are
indicated with numbers. ORFs at both ends of the genome are indicated
with letters. Except for ORFs A and I, which are homologous to 29
proteins 17 and 16.7, respectively, all other ORFs correspond to
polypeptides of unknown function. Predicted transcription termination
sites are indicated with stem-loops. The complete sequence of
the GA-1 genome is in the EMBL/GenBank/DDBJ database under accession
number X96987 (19).
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Identification of the early promoters A1a, A1b, A1c, C2, and
C1b.
Analysis of the 2.8-kb region on the left side of the
GA-1 genome led to the identification of at least three possible
promoters (Fig. 1), all of which contain typical 
35 and 10 boxes
for the 
A RNA polymerase. Promoter A1b is
homologous to the 29 A1 promoter, which is responsible for the
expression of a small RNA (named pRNA) required for the encapsidation
of the viral genome into the proheads (2, 9). The two
other promoters, A1a and A1c, are not present in the genomes of other
related phages whose sequences are known. Promoter A1a is located
upstream of a putative operon containing ORFs M, N, and O, and promoter
A1c is located upstream of another putative operon containing ORFs P,
Q, R, S, and T. These ORFs account for part of the difference in size
of GA-1 DNA and 29 (GA-1 DNA ca. 2 kb larger than 29).
Two other promoters, named C2 and C1b, can be predicted in the right
region of the GA-1 genome. Promoter C2 is homologous
to the
29 C2
promoter that drives the expression of the operon
containing genes 17 and 16.7. In
29, an additional weak promoter,
named C1, is present
within gene 16.7. In the case of GA-1, the
second predicted promoter in
this region maps upstream of gene
16.7. Therefore, this promoter is not
equivalent to
29 C1, and
we have named it
C1b.
To investigate whether the predicted GA-1 promoters correspond to in
vivo promoters, total RNA was purified from infected
cells at different
times after infection. Cells of
Bacillus sp.
strain G1R, the
host for phage GA-1 (
1), were grown in Luria-Bertani
medium (
30) supplemented with 5 mM
MgSO
4 at 37°C to a density
of about 1 × 10
8 cells/ml and infected with phage GA-1 at a
multiplicity of infection
of 5. At different times, samples were taken
and total RNA was
purified as described previously (
20).
Viral transcripts were
detected by primer extension analysis
(
20) using primers hybridizing
at distinct positions
downstream of the predicted transcription
start sites. Promoter A1b,
responsible for the expression of pRNA
(
2), was not
studied because it is homologous to the
29 A1
promoter, which is
known to be actively expressed throughout the
infection cycle
(
20). The expression patterns of the early A2b
and A2c
promoters and the late A3 promoter have been characterized
before
(
11). Analysis of the A3 promoter was included in the
primer extension reactions to serve as an internal control to
mark the
transition from the early to the late stage of transcription.
The
results of the primer extension assays are shown in Fig.
2A.
The relative amounts of the various
transcripts, taking into account
the number of adenines, are presented
in Fig.
2B. Whereas relatively
high levels of transcripts corresponding
to the predicted promoters
A1c and A1a were present early after
infection (5 and 10 min),
these levels were lower at late times. This
indicates that the
A1a and A1c promoters are negatively regulated at
later infection
times. Weaker signals were detected for the transcripts
originating
from the right end of the genome, corresponding to the
predicted
C1b and C2 promoters. Although these promoters are also
negatively
regulated, their expression patterns are different. Whereas
the
transcripts of the A1a and A1c promoters reached their maximum
levels at 10 min postinfection and decreased gradually at later
postinfection times, the maximum level of transcripts for promoters
C2
and C1b was found at 5 min postinfection and decreased quickly
at later
postinfection times.
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FIG. 2.
Characterization of the GA-1 promoters located at both
ends of the genome. (A) Expression of promoters A1c, A1a, C1b, C2, and
A3 throughout the infection cycle. Total RNA was purified from cells at
different times after infection; the transcripts originating from the
indicated promoters were analyzed by primer extension using primers
designed to obtain a cDNA of a distinct length for each promoter. The
A3 promoter, known to be expressed at late times postinfection
(11), was analyzed as a control. Lane A+G corresponds to a
DNA size ladder obtained by chemical sequencing (30). (B)
Quantitative analysis of the transcripts produced from promoters A1c,
A1a, C1b, and C2. The signals obtained for each promoter in the primer
extension reactions (panel A) were quantitated using a laser scanning
densitometer. Since transcripts were detected by the incorporation of
[ -32P]dATP into the cDNA, the signals obtained were
corrected for the number of adenines incorporated into the
corresponding cDNA and normalized relative to an internal control. The
graph shows the amount of mRNA observed through the infection cycle for
each promoter.
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|
The sequences of the newly identified promoters and their transcription
start sites are shown in Fig.
3, together
with those
of the previously characterized GA-1 promoters. All of the
newly
identified promoters, A1a, A1b, A1c, C2, and C1b, have sequences
at their
35 and
10 regions that are very similar to the consensus
promoter sequences recognized by the vegetative RNA polymerase
(
A RNA polymerase) (
16,
21). The
A1b, A1c, and C2 promoters
contain a TG dinucleotide located 1 bp
upstream of the
10 region.
The presence of this so-called "extended
10" motif increases
the strength of
Escherichia coli
promoters (
14,
15,
27)
and of
Bacillus subtilis
phage
29 (
4). The distance between
the
10 and
35
boxes (referred to as the spacer) in each of these
promoters is within
the standard 17 bp ± 1 bp (
16). The absence
of the
extended
10 motif in promoter C1b may, at least in part,
account for
its low level of activity. For the
B. subtilis
phage
29, it has been shown that the
35 region is important for
promoter
activity even in the presence of an extended
10 motif
(
4).
In addition to the extended
10 motif, the sequence
at the
16
region has been shown to be important for promoter strength
in
some
B. subtilis promoters (
35).
The observation that many gram-positive
promoters contain the sequence
TRTG (where R is purine) at the
16 region suggests that this
motif also contributes to promoter
strength. This motif is present at
the GA-1 promoters A1c, A3,
and C2. Inspection of Fig.
3 shows,
however, that a correlation
between the presence or absence of the
10, extended
10,
16,
and
35 motifs and promoter strength is not
always straightforward.
It is known that both DNA sequences and DNA
structure are important
for recognition by the RNA polymerase.
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FIG. 3.
Sequence and transcription start sites of phage GA-1
promoters. Promoters are aligned relative to their 35 boxes. Arrows
indicate the transcription start sites. Those of promoters A1b, A2b,
A2c, and A3 have been reported before (2, 11). The start
site of promoters A1a, A1c, C1b, and C2 were deduced from primer
extension assays of RNA obtained from infected cells. The following are
also indicated: the length (in nucleotides) of the spacer between the
35 and 10 boxes ( denotes that a 35 sequence is not
detected), the presence (+) or absence () of the TG
dinucleotide 1-bp upstream from the 10 box (TGN-10), the relative
strength of each promoter (+++, strong; ++, medium; + weak), and
whether the promoter is active at early or late times postinfection.
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Repression of GA-1 early promoters.
Although the 29 protein
p6 binds DNA with a low sequence specificity, it has a clear preference
for certain DNA regions showing anisotropic bendability
(31). Moreover, under conditions favoring protein-DNA
interactions (high protein concentrations and low ionic strength),
protein p6 can bind, at least in vitro, to the entire 29 genome
(10). The binding of p6 to the ends of the 29 genome
leads to the activation of the initiation of DNA replication and to
repression of the promoters of the right DNA end, C2 and C1 (3,
31). Thus, it was possible that the GA-1 C1b and C2 promoters
could be repressed similarly by GA-1 protein p6. Taking into account
that the A1a and A1c promoters are also repressed from minute 10 after
infection (Fig. 2), we considered that GA-1 protein p6 might also
repress the two latter promoters. Therefore, it was of interest to
determine whether the expression pattern of GA-1 protein p6 was similar
to that of 29. To this end, Bacillus sp. strain G1R was
grown in Luria-Bertani medium (30) supplemented with 5 mM
MgSO4 at 37°C to a density of about 1 × 108 cells/ml and infected with phage GA-1 at a
multiplicity of infection of 5. At different times, samples of 1.5 ml
were taken and centrifuged. The resuspended pellets were sonicated, and
the proteins were resolved in sodium dodecyl sulfate-10 to 20%
polyacrylamide gel electrophoresis gels. As shown in Fig.
4, the amounts of the most abundant early
proteins, p6 and p5, increased during the first 20 min after infection
with GA-1, as occurs in the case of 29. To test the possible role of
protein p6 in transcriptional repression, GA-1 protein p6 was purified
as described previously (6). The activities of the
promoters under study were analyzed by in vitro transcription assays in
the absence or presence of increasing amounts of purified GA-1 protein
p6 and the B. subtilis A
RNA polymerase, purified as described previously (32). In
these experiments, the complete GA-1 genome was used as a template to facilitate the formation of multimeric protein p6-DNA complexes. Each
reaction mixture contained, in a 25-µl volume, 25 mM Tris-HCl (pH
7.5), 10 mM MgCl2, 2 mM dithiothreitol, a 200 µM concentration of each nucleoside triphosphate, 7.5 U of RNasin,
0.2 nM genomic GA-1 DNA (purified as described in reference
13), and 200 nM A RNA polymerase.
Transcripts were analyzed by primer extension assays (20).
Figure 5 shows that promoters A1c, A1a,
C1b, and C2 were repressed in the presence of 20 µM protein p6, while
the early promoter A2b, used as a control, was not; in fact, some activation was observed. The level of repression was different for each
promoter. The activities of the A1c, A1b, C1b, and C2 promoters
decreased to 32, 12, 6, and 5%, respectively. Interestingly, the level
of p6-dependent repression shows a correlation with the expression
profiles observed in vivo (Fig. 2), since transcripts arising from the
A1c promoter were still abundant at late times postinfection, which is
consistent with poor repression. Similarly, transcripts arising from
the A1a promoter were scarce at late times postinfection, which agrees
with efficient repression by protein p6. The parallelism between the
behavior of the promoters in vivo and their response to protein p6 in
vitro suggests that protein p6 represses these promoters in vivo.
Considering that the amounts of protein p6 produced in vivo increase as
infection proceeds, reaching very high concentrations, it is tempting
to speculate that the protein p6 concentration serves to differentially modulate the expression of these early promoters during the infection cycle.
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FIG. 4.
Production of viral proteins during the infection cycle.
Culture samples were obtained at different times after infection of
Bacillus sp. strain G1R with phage GA-1, and proteins
were resolved in sodium dodecyl sulfate-polyacrylamide gels in parallel
with purified GA-1 proteins p5 and p6. The positions of GA-1 proteins
p6 and p5 are indicated.
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FIG. 5.
Effect of GA-1 protein p6 on the expression of GA-1
promoters A2b, A1c, A1a, C1b, and C2 in vitro. Transcription reactions
were performed using the complete GA-1 genome as a template and the
indicated concentrations of GA-1 protein p6. Samples were preincubated
with protein p6 for 10 min at 37°C prior to the addition of RNA
polymerase and nucleoside triphosphates. Transcripts which originated
from each promoter were analyzed by primer extension.
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To further analyze the behavior of GA-1 protein p6 as a repressor, its
ability to repress the GA-1 C2 promoter was studied
under different
ionic strength conditions. It had been demonstrated
that although
29
protein p6 can form a nucleoprotein complex
with the DNA of the right
end of the GA-1 genome, this complex
did not stimulate initiation of
DNA replication in assays containing
the terminal protein and the DNA
polymerase of GA-1 (
6). Therefore,
it was also interesting
to study the effect of this heterologous
DNA-protein p6 complex on the
activity of promoter C2, located
within this region of the genome. The
effects of
29 and GA-1
protein p6 on the GA-1 C2 promoter were
studied in in vitro runoff
transcription assays. Each reaction mixture
contained, in a 25-µl
volume, 25 mM Tris-HCl (pH 7.5); 10 mM
MgCl
2; 2 mM dithiothreitol;
200 µM (each) CTP,
GTP, and ATP; 100 µM [
32-P]UTP (1 µCi);
2 µg of poly[d(I-C)]; 7.5 U of RNasin; 20 nM template
DNA; and KCl
and protein p6 at the concentrations indicated in
the figure legends.
The DNA used as a template was a 246-bp fragment
containing the GA-1 C2
promoter (positions
165 to +81, with respect
to the C2 promoter start
site) and was obtained by PCR from the
viral genome with primers
5'-AAATAGATTCCCCATGAACAAGCG-3' and
5'-GAATAAGGCTAGATAGATATATTTAGG-3'.
Phage
29
protein p6 was purified from
B. subtilis
110NA (
22)
as described previously (
24). The
reaction mixtures were incubated
for 5 min at 37°C, and reactions
were initiated by the addition
of 70 nM
A RNA
polymerase. After an additional incubation for 15 min at
37°C,
reactions were stopped and further processing was carried
out as
described previously (
20). Transcripts were resolved
on
denaturing 6% (wt/vol) polyacrylamide gels and quantified using
a Fuji
BAS-IIIs image analyzer. As shown in Fig.
6, whereas the
GA-1 C2 promoter was
repressed by either phage-encoded p6 protein,
repression by GA-1
protein p6 was in general more efficient than
repression by
29
protein p6. In addition, as predicted, repression
of the GA-1 C2
promoter was more efficient under conditions of
low ionic strength, but
it was also evident at conditions of high
ionic strength.
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FIG. 6.
Capacity of GA-1 and 29 protein p6 to repress the
GA-1 early C2 promoter in vitro. Transcription reactions were carried
out under low (20 mM) (A), medium (100 mM) (B), or high (200 mM) (C)
KCl concentrations. The graphs show promoter activity in the presence
of increasing amounts of protein p6 from either GA-1 (p6G
[open circles]) or 29 (p6 [filled circles]).
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|
In phage
29, the early genes are involved in DNA replication and
transcription regulation. As shown in this work, the GA-1
ORFs that are
absent in
29 are transcribed from early promoters
that are repressed
at late infection times. This suggests that
the putative proteins
encoded by these ORFs may have functions
related to DNA replication
and/or transcription regulation. However,
since both DNA replication
and control of transcription occur
without these ORFs in
29 and
related phages, this could imply
that these putative proteins are
involved in as yet unknown aspects
of these processes. Alternatively,
the possibility that these
ORFs may have a role in interaction with the
infected host cannot
be excluded. Phage
29 infects
B. subtilis, while GA-1 infects
the poorly characterized
Bacillus sp. strain G1R, being unable
to infect intact
B. subtilis cells (
1). Analysis of
the 16S
rRNA of
Bacillus sp. strain G1R (performed by MIDI
LABS Company,
Newark, Del.) showed that it is more than 99%
identical to that
of
Bacillus pumilus, an evolutionary
distance that is small enough
to consider them two strains of the same
species (
33). For comparison,
the similarity with the
B. subtilis 16S rRNA was 94.7%. Therefore,
the
29 and GA-1 hosts are different bacterial species. This may
justify
the need for additional functions in phage GA-1.
| |
ACKNOWLEDGMENTS |
We are grateful to J. M. Lázaro and L. Villar for
protein purification and technical assistance.
This investigation has been supported by research grants 2R01
GM27242-22 from the National Institutes of Health and PB98-0645 from
the Dirección General de Investigación Científica y
Técnica and by an institutional grant from the Fundación
Ramón Areces to the Centro de Biología Molecular
"Severo Ochoa." J.A.H. and W.J.J.M. were supported by postdoctoral
fellowships from the Fundación Raúl González-Salas
and the Spanish Ministry of Education and Culture, respectively.
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FOOTNOTES |
*
Corresponding author. Mailing address: Centro de
Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad
Autónoma, Cantoblanco, 28049 Madrid, Spain. Phone: (34) 91 397 8435. Fax: (34) 91 397 8490. E-mail: msalas{at}cbm.uam.es.
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Journal of Bacteriology, December 2001, p. 6965-6970, Vol. 183, No. 23
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.23.6965-6970.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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