Department of Genetics, Institute of
Molecular and Cell Biology, Estonian Biocentre and Tartu
University, 51010 Tartu, Estonia
The main sigma factor activating gene expression, necessary in
stationary phase and under stress conditions, is 
S. In
contrast to other minor sigma factors, RNA polymerase holoenzyme containing S (ES) recognizes a number of
promoters which are also recognized by that containing
70 (E70). We have previously shown that
transposon Tn4652 can activate silent genes in starving
Pseudomonas putida cells by creating fusion promoters
during transposition. The sequence of the fusion promoters is similar
to the 70-specific promoter consensus. The 
10
hexameric sequence and the sequence downstream from the 10 element
differ among these promoters. We found that transcription from the
fusion promoters is stationary phase specific. Based on in vivo
experiments carried out with wild-type and rpoS-deficient
mutant P. putida, the effect of S on
transcription from the fusion promoters was established only in some of
these promoters. The importance of the sequence of the 10 hexamer has
been pointed out in several published papers, but there is no
information about whether the sequences downstream from the 10
element can affect S-dependent transcription.
Combination of the 10 hexameric sequences and downstream sequences of
different fusion promoters revealed that S-specific
transcription from these promoters is not determined by the 10
hexameric sequence only. The results obtained in this study indicate
that the sequence of the 10 element influences S-specific transcription in concert with the sequence
downstream from the 10 box.
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INTRODUCTION |
In their natural environment, most
bacteria are challenged by widely changing nutrient availability and by
exposure to various forms of physical stress (temperature shock,
oxidative stress, etc.). When starvation or various other stress
factors cause a reduction or cessation of growth, many genes are shut
down while others are induced to help the cells to survive. One way to
modulate gene expression is replacement of the main sigma factor with
an alternative sigma factor that recognizes a specific promoter of the
stimulus response gene (reviewed in reference 19).
In Escherichia coli, S regulates the
expression of more than 100 genes involved in cell survival in the
stationary phase and in response to different stresses (reviewed in
references 14 and 15). The
rpoS gene encoding S has been described also
for nonenteric bacteria, e.g., fluorescent pseudomonads (21, 36,
37, 40). Despite clearly different physiological roles,
S is similar to the major sigma factor 70
in terms of structure and molecular function (7, 26, 42). No
clear differences between 70- and
S-dependent promoters are apparent. A compilation of
S-dependent promoters deduced a 10 consensus sequence,
CTATACT, that is slightly different from the typical
TATAAT sequence recognized by 70
(11). However, the binding patterns of ES and
E70 revealed by DNase I protection experiments are not
completely identical (29, 41). ES appears to
be less dependent on contacts in the 35 region (7, 17,
41). The activity of ES and E70 is
differentially influenced by salt concentrations and by the degree of
negative supercoiling of the DNA template (2, 10, 23).
Additionally, a number of global regulators and histone-like proteins,
such as H-NS, Lrp, CRP, IHF, and Fis, are involved in determination of
sigma factor specificity (29; see also references 14 and 15 for a review and
references cited therein).
We have previously shown the generation of constitutively expressed
promoters in starving population of Pseudomonas putida cells
as a result of base substitutions and deletions and insertion of
Tn4652 (20, 33). These promoters, containing a
sequence similar to the 70-specific promoter consensus,
activated the transcription of phenol degradation genes
pheBA (which encode catechol 1,2-dioxygenase and phenol
monooxygenase, respectively) and enabled bacteria to utilize phenol as
a growth substrate. The fusion promoters were created at the junction
of the sequence of the Tn4652 inverted repeats (provides a
35 hexamer) and the target DNA upstream of the phenol monooxygenase
gene pheA (provides the 10 hexamer of the promoter)
(20, 33) (Fig. 1). Thus, the
sequences of the 10 hexamer and the downstream region of the
promoters are different, depending on the site of the transposon
insertion. In this study, we have shown that the level of transcription
from the six different fusion promoters studied depends on the growth
phase of the P. putida cells, being in all cases higher in
stationary phase. The positive effect of S on
transcription was detected in the case of three fusion promoters. Although the importance of the sequence of the 10 hexamer has been
pointed out in several reports (see references cited above), the role
of the downstream sequences in S-dependent transcription
has not been reported. Analysis of the 10 hexameric sequence
replacement mutant forms of the fusion promoters constructed in this
study indicated that not only the 10 hexameric sequence but also the
sequence downstream from the 10 hexamer is important for
S-dependent transcription.
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FIG. 1.
Nucleotide sequences of fusion promoters PLA1, PRA1,
PRA2, PRA3, PRA4, and PRA7 created at the junction of the sequence of
the Tn4652 inverted repeats and the target DNA upstream of
the phenol monooxygenase gene pheA (33). The 35
hexameric sequence TTGCCT (boxed) of the fusion promoters
originated from either the right (PRA1 to PRA7) or the left (PLA1)
inverted repeat of transposon Tn4652. The sequence of the
10 hexamer (boxed) and the downstream region of the fusion promoters
is different, depending on the site of the transposon insertion. The
70-specific promoter consensus sequences of the 35 and
10 hexamers are shown above the sequences of the fusion promoters.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and growth conditions.
The
bacterial strains and plasmids used in this study are listed in Table
1. E. coli TG1 (6)
was used for the DNA cloning procedures. Exponential- and
stationary-phase cultures of P. putida PaW85 (3)
and derivative strains PKS54 and PKSRpoS (this work) were used for
enzyme assays. We incubated E. coli at 37°C and P. putida at 30°C. E. coli was transformed with plasmid
DNA as described by Hanahan (13). P. putida was
electrotransformed as described by Sharma and Schimke (38).
Bacteria were grown on Luria-Bertani (LB) medium (30).
Antibiotics were added at the following final concentrations:
ampicillin at 100 µg/ml for E. coli; carbenicillin at
1,500 µg/ml, kanamycin at 50 µg/ml, and tetracycline at 10 µg/ml
for P. putida.
Cloning of fusion promoters into a promoter probe vector and
construction of promoter mutants.
For amplification of DNA
fragments containing fusion promoters PLA1, PRA1, PRA2, PRA3, PRA4, and
PRA7 cloned into plasmid pEST1332 upstream of the phenol degradation
genes pheBA (33), two oligonucleotides, PAYC32
(5'-CTCGACCTTTGAGCCAAATG-3') and AB
5'-GGTATGCTTGGCAGTCGT-3'), complementary to sequences
upstream and downstream of the Ecl136II and ClaI
cloning sites in plasmid pEST1332, respectively, were used. PCR
amplification products were cleaved with Ecl136II and
ClaI and cloned into plasmid pBluescript KS(+) restricted
with SmaI and ClaI. Subsequently, the promoters were inserted with BamHI- and XhoI-generated ends
into promoter probe vector pKTlacZ (18).
Mutant promoters L1-R1, L1-R2, L1-R3, L1-R4, and L1-R7 were constructed
by substituting the 10 hexamer of promoter PLA1 for the 10 hexamer
of promoters PRA1, PRA2, PRA3, PRA4, and PRA7, respectively. Mutant
promoters R1-L1, R4-L1, and R7-L1 were constructed by replacing the
10 hexamer of promoters PRA1, PRA4, and PRA7, respectively, with the
10 hexamer of promoter PLA1. Sequences of the mutant promoters are
shown in Fig. 2. For site-directed mutagenesis and amplification of the fusion promoters from pEST1332, oligonucleotide PAYC32 and oligonucleotides containing the specific substitutions were used. The mutating primers were partially
complementary to the sequence at nucleotides (nt) 21 to +7 relative
to the transcriptional start site of the wild-type (wt) promoters. They carried the specific changes within the 10 hexameric sequence, and
the ClaI site was designed 10 to 12 nt downstream of the
10 hexamer. L1Cla was constructed as a control by designing a
ClaI restriction site 12 nt downstream of the 10 hexamer
of PLA1 (Fig. 2). The amplified PCR products were cleaved with
Ecl136II and ClaI and cloned into pBluescript
KS(+). Thereafter, the mutated sequences were inserted upstream of the
lacZ reporter gene into plasmid pKTlacZ by using the
BamHI- and XhoI-generated ends. All introduced
base substitutions were verified by dideoxy sequencing with a Sequenase
version 2.0 kit (Amersham).
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FIG. 2.
Combination of 10 hexamers and downstream sequences of
the fusion promoters. Mutants L1-R1, L1-R2, L1-R3, and L1-R7 were
constructed by replacement of the 10 hexamer of promoter PLA1 with
the 10 hexamer of promoters PRA1, PRA2, PRA3, PRA4, and PRA7,
respectively. In mutants R1-L1, R4-L1, and R7-L1, the 10 hexamers of
PRA1, PRA4, and PRA7, respectively, were replaced with the 10 hexamer
of promoter PLA1. The 10 hexameric sequence is boxed and marked in
bold. All of the mutant promoters carry the ClaI restriction
site (marked in bold and italic) 10 to 12 nt downstream of the 10
hexamer for cloning of the promoter sequences into promoter probe
vector pKTlacZ (19). L1Cla was constructed as a control by
designing a ClaI restriction site 12 nt downstream of the
10 hexamer of PLA1.
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Construction of P. putida PaW85 rpoS
knockout mutant PKS54.
P. putida rpoS knockout mutant PKS54
was constructed as a derivative of PaW85 by interrupting the
rpoS gene with a kanamycin resistance-encoding gene
(Kmr) cloned from transposon Tn903
(34) in plasmid pUC4K (46). The oligonucleotides
used for amplification of the rpoS gene of P. putida PaW85 were designed on the basis of the published sequence of the rpoS gene of P. putida KT2440
(36). Two oligonucleotides, PprpoSall,
(5'-AAAGCTTCCCCTTGCCGGGTGTGTAGAGGA-3') and PprpoSyll (5'-CAAGCGCTGCCAGGGAGAAA-3'), complementary to the sequence
159 nt downstream of the TAG stop codon and 68 nt upstream of the ATG
initiator codon of the rpoS gene of P. putida
KT2440, respectively, were used. The PCR-generated DNA fragment
containing the rpoS gene was subcloned into pBluescript
KS(+) cleaved with EcoRV (pBlcrpoS-I in Table 1). The
EcoRI-generated ends of the DNA fragment containing the
Kmr from plasmid pUC4K were filled with Klenow fragment,
and the blunt-ended DNA segment was inserted into the
Eco72I-cleaved rpoS gene. The resulting
rpoS-Kmr sequence from pBlcrpoS-Kmr
was inserted into conjugative plasmid pUTmini-Tn5 luxAB
(9) by using XbaI and EcoRI sites, and
pUTrpoS-Kmr was selected in E. coli CC118
pir (16). The interrupted rpoS gene was
inserted into the chromosome of PaW85 by homologous recombination using
E. coli S17 pir (31) as the recipient strain.
P. putida rpoS knockout mutant PKS54 was selected at 30°C
on glucose-kanamycin plates and verified with immunoblotting of
P. putida RpoS (see below).
Construction of P. putida rpoS complementation strain
PKS54.
For construction of rpoS complementation strain
PKS54, the rpoS gene was cloned from plasmid pMir61
(36) by using XhoI- and
HindIII-generated ends into the vector pBRlacItac (R. Hõrak and M. Kivisaar, submitted for publication) cleaved with
SalI and SmaI (pBRlacItac-rpoS in Table 1). The
rpoS expression cassette Ptac-rpoS-lacIq was inserted into pUC18Not
(16) using BamHI- and KpnI-generated ends. Thereafter, the hybrid sequence from pUCptac-rpoS was inserted into the NotI site of pUTmini-Tn5 luxAB
(8). The resulting construct, pUTptac-rpoS, was selected in
E. coli CC118 pir (16). The
Ptac-rpoS-lacIq cassette was inserted into the
chromosome of P. putida PKS54 by random insertion using
E. coli S17 pir as the recipient strain. P. putida PKSRpoS was selected at 30°C on
glucose-kanamycin-tetracycline plates. The expression of RpoS in
PKSRpoS was verified with Western blot analysis using polyclonal
antibodies against P. putida RpoS (data not shown).
Cloning, overexpression, and purification of RpoS of P. putida PaW85.
For amplification and cloning of the
rpoS gene of P. putida PaW85, oligonucleotides
RpoSCNco
(5'-TCCCATGG[NcoI]CTCTCAGTAAAGAAGTGCCC-3'; complementary to the sequence 21 nt downstream of the TAG stop codon of rpoS of P. putida KT2440) and RpoSCHind
(5'-GCAAGCTT[HindIII]CTGGAACAATGACTCGCTGGT-3'; complementary to the sequence 20 nt upstream of the ATG initiator codon of rpoS of P. putida KT2440) were used. A
PCR-generated DNA fragment containing the rpoS sequence was
subcloned into pBluescript KS(+) cleaved with EcoRV to
obtain pBlcrpoS-II. Thereafter, the DNA fragment containing the
rpoS gene from pBlcrpoS-II was inserted into pET24d
(Stratagene) with NcoI- and HindIII-cleaved
ends to generate a His tag at the C terminus of RpoS (pET24d-rpoS in
Table 1).
To obtain soluble RpoS-His protein, E. coli BL21(DE3)
(39) carrying pET24d-rpoS was grown overnight at 37°C in
20 ml of M9 minimal medium (1). Subsequently, the culture
was diluted into 500 ml of fresh M9 medium and the bacteria were grown
at 37°C until the optical density of the culture at 590 nm reached about 0.6. For the expression of RpoS-His, the culture was incubated at
20°C for 0.5 h and then induced for 4 h at 20°C by adding
isopropyl-
-D-thiogalactopyranoside (IPTG; final
concentration, 0.5 mM). Cells were pelleted and sonicated in buffer A
(100 mM NaH2PO4, 1 M NaCl, pH 8.0). The cell
lysate was centrifuged at 5,000 × g for 10 min.
Supernatant was loaded onto an Ni2+-iminodiacetic
acid-activated chelating Sepharose 6B column previously equilibrated
with 5 volumes of buffer A. After 4 h of incubation at 10°C, the
loaded column was washed three times with 2 volumes of buffer A
supplemented with 10% glycerol (pH 6.2). The purified His-RpoS was
eluted three times with 2 volumes of buffer A supplemented with 10%
glycerol and 1 M imidazole (pH 6.2). The imidazole and excess salt were
removed by dialyzing the eluate against 1× phosphate-buffered saline,
and the purified protein was stored at 20°C. The purified RpoS was
used for production of mouse anti-RpoS polyclonal antibody for the
immunoblotting assay.
Preparation of cell lysates and immunoblotting of P. putida RpoS.
Cell lysates of P. putida PaW85,
PKS54, and PKS54RpoS were prepared from 50-ml stationary-phase LB
medium cultures and from 200-ml exponential-phase LB medium cultures.
Cells were pelleted and sonicated in 500 µl of 100 mM phosphate
buffer (87 mM Na2HPO4, 13 mM
KH2PO4, pH 7.5). The protein concentration in
cleared lysates was estimated as described by Bradford (5).
Equal amounts (30 µg) of total protein were used for a Western
immunoblotting assay. Proteins were separated by sodium dodecyl
sulfate-10% polyacrylamide gel electrophoresis and transferred to
nitrocellulose membrane (BA85; Schleicher & Schuell). For Western
blotting, the membrane was probed with mouse anti-RpoS polyclonal serum
diluted 1:250, followed by alkaline phosphatase-conjugated coat
anti-mouse immunoglobulin G (LabAS Ltd., Tartu, Estonia) diluted
1:5,000. The blots were developed with
5-bromo-4-chloro-3-indolylphosphate-nitroblue tetrazolium.
-Gal assay.
Exponential- and stationary-phase cells of
P. putida PaW85 and its derivatives harboring different
fusion promoter constructs were used for a -galactosidase (-Gal)
assay performed as specified by Miller (30). The protein
concentration in crude lysates was measured by the method of Bradford
(5). The starting density of the overnight culture for 50 ml
of LB medium at 580 nm was 0.02 (~106 cells/ml). The
samples for the -Gal assay were collected at 4, 12, 24, 36, and
48 h.
| |
RESULTS AND DISCUSSION |
Study of the effect of S on transcription from
fusion promoters.
Promoters were created during transposition of
Tn4652 in stationary-phase cells of P. putida as
fusions between the 35 hexamer provided by the terminal inverted
repeats of Tn4652 and the 10 hexamers in the target DNA
(33). Five of the six different fusion promoters identified
were generated at the junctions of the right terminus of the transposon
and target DNA (promoters PRA1, PRA2, PRA3, PRA4, and PRA7), and in
only one particular case (promoter PLA1) was the 35 hexamer provided
by the left end of Tn4652 (33) (Fig. 1). In this
study, we cloned the sequences containing different fusion promoters
upstream of the reporter gene lacZ in plasmid pKTLacZ
(see Materials and Methods) and studied the effect of the growth phase
of bacteria on transcription from these promoters. P. putida
PaW85 cells carrying lacZ transcriptional fusions were grown
on LB medium, and -Gal activity was measured in both exponentially growing cells (sampled at 4 h) and stationary-phase cells (sampled at 12, 24, 36, and 48 h). The growth curve of bacteria is shown in
Fig. 3A. Data presented in Table
2 demonstrate that the level of
expression of -Gal activity was remarkably elevated in
stationary-phase cells in all cases and it remained high in
deep-stationary-phase cultures during the next 2 days studied. This
indicated that the fusion promoters are certainly stationary phase
specific. Previously we have shown that transcription from the fusion
promoters PRA1 and PLA1, containing sequences of either the right or
left end of Tn4652, respectively, is modulated by IHF and
that the positive effect of IHF becomes apparent in stationary-phase
cells of P. putida (43). However, the fusion
promoters cloned into the pKTLacZ reporter plasmid lacked functional
IHF binding sites but were still expressed at an increased level in
stationary-phase cells.
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FIG. 3.
(A) Growth curve of P. putida PaW85 cells
grown in LB medium. The growth rate of strains PKS54 and PKSRpoS is
similar to that of PaW85. (B) Study of the amounts of RpoS in P. putida PaW85 and PKS54 cells by Western blot analysis with
P. putida anti-RpoS polyclonal antibodies. The cell lysates
used were prepared from P. putida PaW85 cells sampled at h
3, 4, 5, 6, 12, and 24 (lanes 1 to 6). PKS54 cells were sampled at h 24 (lane 7). Purified RpoS-His (lane 8) served as a control. A 30-µg
portion of the total protein from cell lysates and 0.6 µg of RpoS-His
were loaded onto the gel. OD580, optical density at 580 nm.
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TABLE 2.
Study of the expression of fusion promoters in P. putida strain PaW85 and its S-deficient
derivative PKS54
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The most important regulator of stationary-phase-induced genes is sigma
factor S, encoded by rpoS, which directs the
expression of nearly 100 genes in E. coli (25,
45) and is considered the second principal sigma factor
(42). The requirement of S for stimulation of
transcription has also been described in the case of genes originating
from P. putida (27, 28, 32). The gene homologous
to the rpoS gene of E. coli has been cloned from P. putida by complementation of the
rpoS-deficient E. coli strain (36). In
order to test whether functional S would be responsible
for the increased level of transcription from the fusion promoters in
stationary-phase cells of P. putida, we constructed an
rpoS knockout mutant of P. putida. P. putida PaW85 rpoS knockout mutant PKS54 was obtained by homologous
recombination between the P. putida original chromosomal
rpoS gene and the rpoS gene interrupted by a
kanamycin resistance gene. P. putida S was
overexpressed using protein expression vector pET24d and purified to
obtain polyclonal antibodies against it (for details, see Materials and
Methods). The absence of S in the mutant strain was
verified by Western blot analysis (Fig. 3B). In E. coli, an
increase in the cellular S concentration during entry
into stationary phase has been observed (12, 24, 42).
Western blot analysis of the intracellular S content of
P. putida (Fig. 3B) also demonstrated that a low level of
this sigma factor is detectable even in actively growing cells (h 3 and
4) and that the intracellular level of S increases in
bacteria sampled from the late-exponential-phase culture (h 5 and 6).
We compared the expression of the fusion promoters in wt strain PaW85
and in its S-deficient derivative PKS54 (Table 2). The
effect of S became evident only in the case of three
fusion promoters: the level of transcription from promoters PLA1, PRA4,
and PRA7 was approximately three times lower in strain PKS54 than in
the wt strain. The maximal difference appeared in a
deep-stationary-phase culture (cells sampled at 24 h and later).
At the same time, the level of transcription from fusion promoters
PRA1, PRA2, and PRA3 did not depend on the presence or absence of
functional S in the cells. In order to test whether the
full level of transcription from fusion promoters PLA1, PRA4, and PRA7
would be restored by complementation of the S-deficient
mutant with the functional rpoS gene, strain PKSRpoS was
constructed. In this strain, the rpoS gene was introduced into the chromosome of PKS54 under control of the Ptac
promoter and the lacIq repressor (Materials and
Methods). As a result of complementation, the level of expression of
fusion promoters PLA1, PRA4, and PRA7 in PKSRpoS was approximately the
same as that estimated in the wt strain (data not shown).
As shown in Table 2, the stationary-phase-induced transcription from
the fusion promoters cannot be explained as the effect of
S only. The S-independent increase in
transcription became evident with all of the fusion promoters. Gel
mobility shift experiments with Tn4652 ends and crude lysate
of P. putida have revealed that in addition to IHF, some
other proteins form specific complexes with the ends of
Tn4652 (18, 43). The amount of the protein-DNA
complexes detected depends on the growth phase of the bacteria used for the preparation of cell extracts (43). It is therefore
possible that a protein(s) which binds termini of Tn4652
could sequester transcription from the fusion promoters in a growth
phase-dependent manner. Experiments intended to identify these factors
are currently in progress.
Study of the effect of combination of the 10 hexameric sequences
and downstream sequences of the fusion promoters on transcription in
S-deficient P. putida.
Notably, the fusion
promoters providing a lower level of reporter gene expression in the
rpoS mutant strain differed from the others only by the 10
hexameric sequence and by the sequence downstream from that. There was
only one mismatch between the right and left inverted repeat sequences,
making the spacer region of the fusion promoters created from either
the right or the left end of the transposon different by 1 nt (Fig. 1).
Thus, our results indicated that the sequence of the 10 element could
be important for S-dependent transcription. The same has
also been concluded in other published reports (17, 22, 41).
A compilation of S-dependent promoters deduced a 10
consensus sequence, CTATACT (11). Fusion
promoters PLA1, PRA4, and PRA7, that were expressed at a decreased
level in the rpoS-deficient background, contained 10
hexamers TATACT, TAAACT, and TATAAT,
respectively, that were preceded by a C nucleotide. At the same
time, the sequence of the 10 element of S-independent
promoter PRA1 differed in two position from the 10 hexamer of PLA1
(sequence TATCAT instead of TATACT).
To confirm whether the S-dependent transcription from
promoters PLA1, PRA4, and PRA7 would be influenced by the specific
sequence of the 10 hexamer, the identical downstream sequence was
constructed for all of the different fusion promoters studied. For that
purpose, the 10 sequence of PLA1 (TATACT) was replaced
with the sequences of the 10 hexamers of the other fusion promoters
(Fig. 2). The sequence of PLA1 was chosen because its 10 hexamer is
identical to the fic promoter 10 element (42,
44). S-dependent transcription initiation from the
fic promoter requires the 10 hexamer TATACT but
does not need a specific sequence in the 35 region (17).
The resulting PLA1 10 substitution mutants L1-R1, L1-R2, L1-R3,
L1-R4, and L1-R7 contained 12 nt of the downstream sequence of PLA1 and
an artificial ClaI site for cloning of these promoters into
the pKTLacZ vector. The same strategy was used to subclone PLA1
upstream of the lacZ gene to obtain L1Cla. We also replaced
the 10 hexamer of promoters PRA1, PRA4, and PRA7 with the 10
sequence of PLA1 (see R1-L1, R4-L1, and R7-L1 in Fig. 2). -Gal
activities measured in the wt strain and in its rpoS-deficient derivative PKS54 carrying different 10
substitution mutants revealed unexpected results (Table
3). Although the expression of fusion
promoters PRA1 and PRA3 was not influenced by S in the
original location of these sequences, a two- to fourfold positive
effect of S was still apparent when the 10 hexameric
sequence of PLA1 was replaced with 10 hexamers of PRA1 and PRA3. At
the same time, only a very mild effect, if any, was observed in the
case of L1-R7 despite the fact that both PLA1 and PRA7 separately
exhibited decreased expression in the rpoS mutant strain. No
effect of the presence of functional S was detected in
the case of L1-R2. A mild effect, not exceeding 1.5 times, became
evident in R1-L1 and R7-L1 (the 10 hexamers of PRA1 and PRA7 were
replaced with the 10 hexamer of PLA1, respectively), and an
approximately twofold effect was revealed when the 10 element of PRA4
was changed to that of PLA1 (see R4-L1).
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TABLE 3.
Study of the expression of mutant fusion promoters in
P. putida strain PaW85 and its S-deficient
derivative PKS54
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To summarize the results obtained in this study, we can conclude that
neither the sequence of the 10 hexamer of the fusion promoters nor
the sequence downstream of the 10 element can separately affect
S-dependent transcription. Rather, the sequence of the
10 hexamer in concert with the sequence downstream from the 10
element influences S-dependent transcription from the
fusion promoters. Based on in vivo experiments carried out in this
study, we cannot elucidate the exact mechanisms of interaction of the
ES holoenzyme with downstream sequences of promoters
during transcription initiation; i.e., it is difficult to say whether
the downstream sequences could somehow be involved in
ES-specific promoter recognition. Transcription
initiation involves two major steps, formation of a closed complex
which, in a second step, isomerizes to an open complex (47).
Until now, in contrast to E70, only a few biochemical
studies on the stationary-phase RNA polymerase have been performed.
Comparative investigations have revealed that for specific DNA-protein
binding, the ES holoenzyme interacts with a smaller part
of the promoter than E70 and recognizes the sequence in
the 10 region (17, 41). Recently, the interaction of
S and 70 in RNA polymerase-promoter open
complexes was analyzed using FeBABE nuclease (4, 7, 35). The
two holoenzymes revealed similar cutting patterns, but the cutting
pattern of ES extended toward the downstream part of the
promoter, around +10 and +20 (7). Therefore, taking together
the published data and the results presented in this study, we propose
that the possible role of downstream sequences in
S-dependent transcription needs more consideration.
We thank M. I. Ramos-Gonzales for kindly providing plasmid
pMir61 and R. Hõrak for construction of P. putida
strain PKSRpoS. We also thank T. Alamäe and R. Hõrak for
critically reading the manuscript.
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10 Hexamers
and Downstream Sequences on Stationary-Phase-Specific Sigma Factor
S-Dependent Transcription in Pseudomonas
putida
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Abstract
Introduction
