From the Department of Biochemistry and Molecular Biology, Saitama University, Urawa 338-8570, Japan
Received for publication, February 23, 2001, and in revised form, April 23, 2001
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ABSTRACT |
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A novel heat shock gene,
orf7.5, which encodes a putative acidic polypeptide
of 63 amino acids, was cloned from the cyanobacterium Synechococcus sp. PCC 7942. Northern blot analysis revealed
the presence of 400- and 330-base orf7.5 mRNAs,
which were barely detectable in the cells grown at 30 °C but
increased transiently in response to heat shock at 40 or 45 °C.
Primer extension analysis showed that the two mRNAs have different
5'-ends. Chloramphenicol enhanced the accumulation of the
orf7.5 mRNA, whereas it inhibited the increase
in the amount of the groESL mRNA. To reveal the role of the orf7.5 gene in thermal stress management, we
constructed a stable mutant in which a gene conferring resistance to an
antibiotic was inserted into the coding region of the
orf7.5 gene. The interruption led to a marked
inhibition of growth at 45 °C and a decrease in the basal and
acquired thermo-tolerances at 50 °C in the transformants, indicating
that the gene plays a role in thermal stress management. The
orf7.5 mutant could be complemented with a return to
the wild type phenotype by a DNA fragment containing
orf7.5 but not by mutated
orf7.5s, in which a nonsense mutation was
generated by introducing a frameshift or a point mutation within the
orf7.5-coding region. Thus, thermo-tolerance
requires an appropriate translation product, not simply a
transcript. Accumulation of the groESL transcript in the
orf7.5 mutant was strongly reduced, suggesting that
the orf7.5 gene product controls the expression of
the groESL operon.
Cyanobacteria are photoautotrophic prokaryotes that are
phylogenetically and physiologically related to the chloroplasts of photosynthetic eukaryotes. Thus, cyanobacteria, especially
transformable ones such as Synechocystis sp. PCC 6803 and
Synechococcus sp. PCC 7942, have proven to be valuable model
organisms to elucidate chloroplast functions such as photosynthesis.
Cyanobacteria, like other organisms, synthesize a diverse range of heat
shock proteins (Hsps)1 upon
exposure to high temperatures (1-4). To prove a specific contribution
of a Hsp in thermo-tolerance in cyanobacteria, disruption mutants of
the clpB, hsp16.6, and htpG genes have
been constructed (5-7). Interestingly, all these disruptants showed
much more striking thermo-sensitive phenotypes than those of
Escherichia coli or other prokaryotes, suggesting that those
Hsps have a particularly important role for photosynthetic prokaryotes.
In fact, photosynthetic oxygen evolution activity in the
clpB and hsp16.6 mutants was more susceptible to
high temperature inactivation than that in the wild type (5, 6).
The regulation of the expression of cyanobacterial heat shock genes
remains poorly understood. Webb et al. (3) identify sequences upstream of the transcription start site of the
groESL gene from Synechococcus sp. PCC 7942 cells
that were similar to the consensus heat shock promoters of E. coli recognized by sigma factor 32. However, there is no evidence
for the presence of sigma factor 32 in cyanobacteria. As far as we
know, there is no report that an alternative sigma factor may control
the heat shock regulon in cyanobacteria. The nine-nucleotide inverted
repeat sequence called CIRCE (8) is a regulatory element of heat
shock induction that is conserved in cyanobacteria as well as other
prokaryotes. The CIRCE element has been reported to be present around
the transcription start site in groESL and/or
dnaK operons of more than 30 different bacterial species,
and it is thought to be an operator with which the HrcA repressor
protein interacts (9-11). CIRCE has been identified upstream of
groEL genes in cyanobacteria (12-14). Recently, it was
shown that heat shock strongly enhanced accumulation of transcripts of
the two groEL genes, groEL and cpn60,
in Synechocystis PCC 6803 in the light, but induction was
lower in the dark (14). Light appears to exert its effect through the
photosynthetic electron transport since DCMU, an inhibitor of electron
transport, suppressed the accumulation of transcripts of the two
groEL genes in the light. Those results suggest that
cyanobacteria may have evolved a unique regulatory mechanism to induce
the groEL genes. Evidence for additional regulatory
mechanisms came from an inspection of promoter sequences. The potential
transcription initiation site of the hspA gene, encoding a
small Hsp homologue in the thermophilic cyanobacterium
Synechococcus vulcanus, was preceded by typical vegetative
promoter sequences, although its transcript was clearly heat-inducible
(15). There was no CIRCE element around the potential transcription
initiation site of the hspA gene, indicating that an unknown
regulatory mechanism suppresses the expression of hspA in
cyanobacteria under non-heat shock conditions. Furthermore, the gene
appears to be regulated post-transcriptionally because its mRNA was
more stable at a heat shock temperature 63 °C than at 50 °C
(15).
In this paper, we report the isolation and characterization of a novel
heat shock gene, orf7.5, from
Synechococcus sp. PCC 7942. To reveal the function of the
orf7.5 gene, we generated a mutant strain in which
orf7.5 was insertionally disrupted by gene targeting.
Analysis of the mutant showed the involvement of the
orf7.5 gene in the growth and survival of
Synechococcus sp. PCC 7942 under high temperatures and,
additionally, in the expression of the groESL operon.
Organisms and Culture Conditions--
Unless otherwise
indicated, Synechococcus sp. PCC 7942 cells were grown at
30 °C under a light intensity of 30 µE/m2/s in liquid
BG-11 medium (16) or on BG-11 plates containing 1.5% (w/v) agar and
0.3% (w/v) sodium thiosulfate. The BG-11 was modified to contain 5 mM TES-NaOH, pH 8.0, and 50 mg/ml
Na2CO3. The liquid culture was bubbled with air.
DNA Sequencing--
Sequencing single-stranded DNA was carried
out by using an AutoRead sequencing kit (Amersham Pharmacia Biotech)
and a DNA sequencer (DSQ-1, Shimadzu, Kyoto, Japan). The nucleotide
sequences were aligned and analyzed using GENETYX software (Software
Development Co. Ltd., Tokyo, Japan).
Probe Preparation and Genomic Library Screening--
The
orf7.5 gene from Synechococcus sp. PCC
7942 was found during the amplification of a small Hsp gene by
polymerase chain reaction (PCR). One of the PCR primers,
5'-GG(A/G)CCITT(C/T)(C/A)GIGA(A/G)GA-3', was based on the
amino-terminal sequence of HspA of S. vulcanus (15),
EPFREED, and the other primer, 5'-ATIC(GT)IA(GA)IGTIA(GA)IACICC-3', was
based on the internal amino acid sequence of the HSP18 of Streptomyces albus (17), GVLTLRI. A 300-bp fragment was
amplified from the Synechococcus sp. PCC 7942 genome after
90 cycles of denaturation for 1 min at 94 °C, annealing for 1 min at
55 °C and extension for 2 min at 72 °C, although nothing was
produced after 30 PCR cycles. The product was designated as PCR-1. The PCR-1 was cloned into pT7Blue T-vector (Novagen, Madison, WI).
PCR-1 was labeled with [ Isolation of Genomic DNA from Synechococcus sp. PCC 7942 and
Southern Blot Analysis--
Southern blot analysis was performed as
described previously (19) except for the following procedure. A 276-bp
NcoI fragment (Fig. 1A) containing the
orf7.5 gene was labeled as described above and used
as a probe for the hybridization. After hybridization, the membrane was
washed in 2× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate) containing 0.1% (w/v) SDS and then 1×
SSC containing 0.1% (w/v) SDS at 65 °C for 30 min each.
Preparation of Total RNA from Synechococcus sp. PCC 7942 and
Northern Blot Analysis--
Total RNA from Synechococcus
sp. PCC 7942 was prepared as described previously (19) from cells
incubated for different time intervals after shifting cultures from 30 to 40 or 45 °C. During the incubation, the culture was illuminated
and air-bubbled as described above. Unless otherwise indicated, 10 µg
of total RNA was electrophoresed on a denaturing 1.5% (w/v) agarose
gel containing 6.6% (w/v) formaldehyde. Northern blotting and
hybridization with the 32P-labeled 276-bp NcoI
fragment (Fig. 1A) as a probe were performed as described
previously (19). After hybridization, the membrane was washed in 6×
SSC at 65 °C for 60 min. The size of the mRNA was determined
using an RNA ladder (Life Technologies, Inc.). The hybridization
signals were detected with a BAS1000 Mac bio-imaging analyzer (Fuji
Film, Tokyo, Japan).
In Fig. 9, the orf7.5, groEL, and
htpG mRNAs were detected by hybridization with
digoxigenin-labeled RNA probes as instructed by the manufacturer (Roche
Molecular Biochemicals). A suitable fragment for the preparation of an
orf7.5-specific probe was generated by cloning a
0.8-kbp DNA fragment (Fig. 1A) containing the
orf7.5 gene into the multicloning sites,
EcoRI and PstI, of pBluescript II KS (+). The
fragment (nucleotides 9-820, Fig. 1B) was amplified by PCR
with primers
(5'-AGGTTGAATTCGTGGGCACAGCAA-3' and 5'-TTCAACCTGCAGCCAGGTTT-3', where A and
T are mutations to create an EcoRI
restriction site), and the cloned 5.0-kbp
XhoI-EcoRI fragment was described above as a
template. The resulting plasmid, pBS-EP, was used for the in
vitro production of a digoxigenin-labeled,
orf7.5-specific RNA probe with T7 RNA polymerase
after linearization with EcoRI. For the preparation of a
groEL-specific probe, a 1.1-kbp
EcoRI-HincII fragment containing the 3' half of
groES and the 5' half of groEL of S. vulcanus (13) was cloned into pBluescript II KS (+). The resulting
plasmid was digested with EcoRI, and the linearized DNA was
used for the in vitro production of a digoxigenin-labeled
groES and groEL-specific RNA probe with T3 RNA
polymerase. For the preparation of a htpG-specific probe, a
3.6-kbp EcoRI fragment containing the htpG gene
of Synechococcus sp. PCC 7942 (7) was cloned into
pBluescript II KS (+), linearlized with BglII, and used for
the in vitro production of a digoxigenin-labeled RNA probe
with T3 RNA polymerase. The RNA polymerase transcribed the 0.45-kbp
BglII-EcoRI region of the htpG gene
(7).
Primer Extension Analysis--
Primer extension analysis was
performed according to Sambrook et al. (18). A synthetic
oligonucleotide, 5'-TCGTAGAGGTTACCGAACGC-3', which is complementary to
the region downstream of the initiation codon of
orf7.5 (nucleotides 500-519, see Fig. 1B)
was labeled with [ Reverse Transcription-PCR--
Reverse transcription-PCR was
performed using GeneAmp thermostable rTth reverse transcriptase RNA PCR
kit as directed by the manufacturer (Roche Molecular Biochemicals).
Total RNA was prepared as described above, and contaminating DNA was
removed by S.N.A.P. total RNA isolation kit (Invitrogen,
Carlsbad, CA). The antisense oligonucleotide primer
(5'-TATCTTACGGCTTTGGCCGA-3') complementary to nucleotides 693-712
(Fig. 1B) was used for cDNA synthesis that was performed
at 60 °C for 30 min. PCR amplification was performed with a sense
primer
(5'-TAGGGAATTCCACCCTAGTTGC-3', where G and T are a
mutation and an insertion, respectively, to create an EcoRI
restriction site) corresponding to nucleotides 288-309 (Fig.
1B) under the cycling conditions of 35 cycles of denaturation for 30 s at 94 °C, annealing for 30 s at
60 °C, and extension for 1 min at 72 °C and the final extension
for 7 min at 72 °C.
Construction of a Mutant--
A 2.0-kbp
HindIII/EcoRI chromosomal fragment (Fig.
1A) cloned in pBluescript II KS (+) was digested at the
unique BstEII site in orf7.5. A
kanamycin-resistant gene cassette, which was isolated by digesting
pUC4K (Amersham Pharmacia Biotech) by BamHI, was inserted
into the restriction site in the forward direction (the same gene
orientation as that of orf7.5). The resulting plasmid was named pNT31 (Fig. 5). The construct was used to transform cells of
naturally competent Synechococcus sp. PCC 7942 through homologous recombination. These cells were segregated for a few generations by single colony selection on BG-11 agar plates to isolate
mutant strains (20). 20 µg/ml kanamycin sulfate was used for
selection of transformants. One of the clones was designated as NT31.
Viability Assays--
The Synechococcus sp. PCC 7942 cells were grown to mid log phase at 30 °C under a light intensity
of 35 µE/m2/s as described above and then diluted to an
absorbance of 0.50 at 730 nm. For basal thermo-tolerance assays, 20 ml
of the diluted culture was incubated at 50 °C for 20 min. Aliquots
of the culture taken before and after the high temperature treatment
were serially diluted (5 times each) in fresh, sterile BG-11 medium. A
10-µl aliquot from each dilution was then spotted onto a BG-11 plate, and the culture was grown at 30 °C under a light intensity of 35 µE/m2/s for a week. Cell survival was determined by
counting the number of colonies in the most and second-most diluted
cultures and multiplying their average by the appropriate dilution
factor. At least three independent replicate experiments were carried
out for all temperature experiments. Acquired thermo-tolerance assays
were performed as above, except 20 ml of the diluted culture was given
a pretreatment at 42 °C for 60 min before the high temperature
treatment at 50 °C for 20 min. In both assays, the cultures were
continuously bubbled with air, and the photon irradience was maintained
at 35 µE/m2/s throughout the heat treatments at 42 or
50 °C.
Complementation of NT31 with a DNA Fragment Containing
orf7.5--
pBS-EP, which was used for the preparation of an
orf7.5-specific RNA probe, was digested with
XbaI and XhoI at the cloning sites of the vector
and subcloned into pAM1573 to generate plasmid pNI52. pAM1573, which
was provided by Professor Susan S. Golden (Texas A & M University,
College Station, TX), has the cloning sites and a chloramphenicol
resistance gene inserted into a segment of DNA (termed as a neutral
site) from the Synechococcus sp. PCC 7942 chromosome. The
neutral site mediates homologous recombination with the chromosome
without causing any apparent phenotype. pNI52 was introduced into
Synechococcus sp. PCC 7942 as described above, and
transformants were selected on BG-11 agar plates containing 10 µg/ml
kanamycin and 7.5 µg/ml chloramphenicol. One of the transformants was
designated as NI52.
Site-directed Mutagenesis of the orf7.5 Gene--
A
frameshift or a point mutation within the
orf7.5-coding region was generated by QuikChange
site-directed mutagenesis kit (Stratagene). Two primers
(5'-GCGTTCGGTAACCTCTGACGAAATCTTTGGGCG-3', 5'-CGCCCAAAGATTTCGTCAGAGGTTACCGAACGC-3')
containing an insertion of a single base at the ninth codon of
orf7.5 (underlined bases in the above sequences, see
also Fig. 8A), each complementary to opposite strands of the
vector pBS-EP, were extended by PCR. Then the product was treated with
DpnI to digest the parental template pBS-EP and to select
for mutation-containing synthesized DNA. The DNA incorporating a
frameshift in orf7.5 was designated as pNI71. In the
same way, pNI81 in which a point mutation was incorporated at the third
codon of orf7.5 (underlined bases in the following
sequences, see also Fig. 8A) was generated using two
primers, 5'-GAGGATCCCATGGAGTAAGCGTTCGGTAACC-3',
5'-GGTTACCGAACGCTTACTCCATGGGATCCTC-3'. The
mutations were confirmed by sequencing orf7.5 in
pNI71 and pNI81. Either pNI71 or pNI81 was introduced into
Synechococcus sp. PCC 7942 as described above, and
transformants were selected on BG-11 agar plates containing 10 µg/ml
kanamycin and 7.5 µg/ml chloramphenicol. One of the clones from each
transformation was isolated and designated as NI71 and NI81, respectively.
Nucleotide Sequence Accession Number--
The sequence data
reported here (see Fig. 1) will appear in the DDBJ, EMBL, and
GenBankTM nucleotide sequence data bases with the accession
number AB002694.
Cloning and Sequencing of Two Novel Open Reading Frames and
purB from Synechococcus sp. PCC 7942--
Initially, we intended to
amplify a small Hsp gene from the unicellular cyanobacterium
Synechococcus sp. PCC 7942 by PCR with degenerate primers to
small Hsps from other prokaryotes. Although there was no product after
30 cycles of polymerase reactions, further-extended PCR resulted in a
0.3-kbp DNA fragment. The sequence of the DNA fragment does not reveal
close relatedness to any gene in the data base. We further examined the
product to see if it is a portion of a heat-inducible gene since we
have embarked on a project to identify cyanobacterial Hsps and their
genes (7, 13, 15, 21). Northern blot analysis revealed transient
increases during heat shock of 400- and 330-base RNAs with which the
0.3-kbp PCR product hybridized (data not shown). These results
indicated that the PCR product constitutes a novel heat shock gene.
Thus, we went on to clone a DNA fragment containing the whole gene by screening a Synechococcus genomic library with this PCR product.
There were three open reading frames (orfs) in the
sequenced region of 2.4 kbp (Fig.
1A). We could not find any
nucleotide or amino acid sequences in Cyanobase (data base on the
genome of cyanobacterium Synechocystis sp. PCC 6803)
that exhibits significant homology to the smaller orfs. One
of them, designated as orf14.1 (the number is based on the
estimated molecular weight for the orf) is located from
bases 333 to 713 of the sequence shown in Fig. 1B. Within
orf14.1, there was another orf that we designated as orf7.5. The nucleotide sequence of the PCR product
corresponded to a region from bases 478 to 607 of the sequence shown in
Fig. 1B. The latter half of the PCR product did not
correspond to the sequence. Thus, the 400- and 330-base RNAs described
above are likely to be transcripts of orf14.1 and/or
orf7.5. The amino acid sequence deduced from the
larger orf, which is located 54-bp downstream of
orf14.1, showed significant homology to adenylosuccinate
lyase (PurB) of Bacillus subtilis (22).
The number of orf14.1 and orf7.5
copies in the Synechococcus sp. PCC 7942 genome was
determined by Southern blot analysis. Synechococcus sp. PCC
7942 genomic DNA digested with EcoRI, HincII, HindIII, PstI, SalI, or
XhoI was hybridized using the radiolabeled 276-bp
NcoI DNA fragment (shown as a dotted line in Fig.
1A) as a probe. The probe hybridized to only one DNA
fragment for each restriction endonuclease, demonstrating that there is
no other gene in the genome that is extensively homologous to
orf14.1 and orf7.5 (data not shown).
Heat Induction of orf14.1 and/or orf7.5--
To
confirm that orf14.1 and/or orf7.5 is
induced by heat shock, we performed Northern blot analysis with total
RNA isolated from cells after a shift from 30 to 40 °C. The
NcoI DNA fragment containing a 3'-portion of
orf14.1, whole orf7.5, and upstream untranslated region of the purB gene hybridized with 400- and 330-base RNAs (Fig. 2A).
The 400- and 330-base RNAs, barely detectable at 30 °C, increased 4- and 3-fold, respectively, after a 15-min exposure to 40 °C and then
diminished during 45 min of prolonged incubation (Fig. 2A).
The Northern blot was stripped and reprobed with the
32P-labeled psaD gene of
Synechocystis sp. PCC 6803, which encodes one of the
peripheral subunits of photosystem I (23). The psaD gene was
shown previously not to be heat-induced in Synechocystis sp.
PCC 6803 (24), thus serving as a control for a heat-induced gene. The
level of the psaD mRNA remained unchanged during the heat shock (Fig. 2C), confirming that the two RNAs are
heat-induced. Similar experiments were carried out with total RNA
isolated from cultures heat-shocked at 45 °C. The accumulation of
the 400- and 330-base RNAs was 9- and 4-fold, respectively, after a
15-min exposure to 45 °C (Fig. 2B). Therefore, the
400-base RNA increased further at 45 °C. Northern blot analysis with
an RNA probe whose sequence is complementary to orf14.1 and
orf7.5 resulted in similar results (Fig. 9). These
results suggested that orf14.1 and/or orf7.5 were induced upon heat shock treatment.
The DNA and RNA fragments sused as probes contain sequences beyond
orf14.1 and orf7.5. Therefore, the probes
could be hybridizing to an RNA downstream of the orfs. We
performed Northern blot analysis with the radiolabeled
SacI-EcoRV DNA probe (see Fig. 1A) to
examine expression of purB. No RNA hybridizing with the
purB probe in total RNA that was prepared from cells grown
at 30 °C or heat-shocked at 45 °C was detected (data not shown).
Thus, it appears that the putative purB gene is not
expressed significantly under both normal and stress conditions. The
results also confirm that the 400- and 330-base mRNAs were the
transcripts of orf14.1 and/or orf7.5.
The 400- and 330-base mRNAs Were the Transcripts of
orf7.5--
The two RNA species of 400 and 330 bases may be
transcripts of orf14.1 and orf7.5,
respectively. Alternatively, both mRNAs originate from the same
orf. To clarify the origin of the RNAs, we determined the
5'-ends of the 400- and 330-base mRNAs by primer extension analysis
using total RNA and a synthetic oligonucleotide primer complementary to
the 5'-end of orf7.5. Total RNA was isolated from
Synechococcus sp. PCC 7942 cells grown at 30 °C or cells heat-shocked at 45 °C for 15, 30, 60, and 120 min. The analysis revealed two extended products (Fig. 3).
No more extended products were detected within 200 bases upstream of P1
(data not shown). The result indicated that the two mRNAs have
different 5'-ends that correspond to the P1 and P2 positions of the
genomic DNA shown in Fig. 1B. The number of both transcripts
increased with the temperature shift from 30 to 45 °C, although the
transcripts initiated at P1 increased much more than those initiated at
P2. P1 and P2 were located 141 and 71 bases upstream of the ATG start codon of orf7.5, respectively (Fig. 1B and
Fig. 3). Since P1 and P2 are located within orf14.1 (Fig.
1A), the two mRNAs are likely to be the transcripts of
orf7.5. We could find neither the E. coli-type heat shock promoter sequences recognized by sigma factor 32 (25) nor the CIRCE (8) sequence upstream of P1 and P2. Instead, P1
was preceded by sequences that resemble the consensus sequences of
promoters recognized by the vegetative sigma factors of B. subtilis as well as that of E. coli (Fig.
1B, underlined) (25). We could not find any
promoter sequences preceding P2. P1 was located 70 bases upstream of P2
(Fig. 1B), supporting the conclusion that the 400- and 330-base RNAs (Fig. 2B) have the same 3'-end.
We confirmed by reverse transcription-PCR that transcription of
orf14.1 must be negligible even if it is actually expressed. There was only a negligible, if any, amount of PCR products with the
expected size (0.43 kbp) when a primer corresponding to the sequence
41-bp upstream of P1 and a primer complementary to the 3'- end of
orf14.1 were used for the PCR reaction to amplify a DNA
fragment complementary to the orf14.1 mRNA (data not
shown). In addition, we constructed a mutant in which an antibiotic
resistance gene was inserted into a site located 38 bases upstream of
the first ATG codon of orf14.1. The insertion did not cause
any apparent phenotype at both 30 and 45 °C. Thus, we conclude that
orf14.1 does not function as an expressed gene.
Differential Effect of Chloramphenicol on the Expression of the
groESL Operon and orf7.5--
There is no CIRCE regulatory
element around the orf7.5 transcription start site,
whereas the groESL operon has one. Thus, we thought that
there should be some difference in the heat induction of the two genes.
Previously, we showed the protein synthesis inhibitor chloramphenicol
completely inhibited the accumulation of mRNA from the
groESL operon in S. vulcanus when added before the heat shock (13). This suggested the involvement of heat-induced production of a protein for the expression of the operon. We attempted to determine whether the antibiotic may exert a different effect on the
expression of the orf7.5 gene. When chloramphenicol
was added before the shift from 30 to 45 °C, the accumulation of the 400- and 330-base RNAs continued during the heat shock (Fig.
4A). The increase in the level
of mRNA was remarkable. These results were in contrast with the
transient increase of the mRNA accumulation in the control (Fig.
4A). The Northern blot was stripped and reprobed with a
32P-labeled groEL gene from S. vulcanus (13). As opposed to the increased accumulation of the
400- and 330-base RNAs in the control, that of the groESL
mRNA was completely inhibited by the addition of chloramphenicol
(Fig. 4B).
Disruption of orf7.5--
To elucidate the function of the
orf7.5 gene in relation to thermo-tolerance, the gene
was disrupted by inserting a kanamycin-resistant gene cassette into the
BstEII site located 19 bp downstream of the initiation codon
of orf7.5. The mutant was designated as NT31 (Fig.
5).
Integration and complete segregation of the orf7.5
interruption construct within the mutant genome was confirmed by
Southern blot analysis and PCR analysis (data not shown). The size and the number of the restricted DNA fragments hybridized with the probe
and the PCR-amplified products were as expected when the kanamycin-resistant gene cassette (1.3 kbp) was introduced into the
loci shown in Fig. 5 in all the copies of the mutant chromosome. 400- and 330-base RNAs were not detected in the mutant cells (Fig. 9). These
results indicate that orf7.5 is dispensable for the cyanobacterial growth under the normal conditions.
Role of orf7.5 in Thermo-tolerance--
We examined whether
the interruption of orf7.5 has any effect on the
thermo-tolerance of Synechococcus sp. PCC 7942. The growth rates of the wild type and the mutant, NT31, were measured at 30 or
45 °C by monitoring the apparent absorbance of the cultures at 730 nm. The growth rate of the mutant was similar to that of the wild type
at 30 °C (Fig. 6). However, when the
temperature was increased to 45 °C, the mutant NT31 stopped growing
after a short initial growth period of ~20 h (Fig. 6), which almost equals the time required for one cell division. The wild type could
grow at this temperature. The temperature-sensitive phenotype of NT31
showed that orf7.5 plays an important role in
cyanobacterial growth even at moderately high temperature.
We attempted to determine whether there is any difference in basal
thermo-tolerance between the wild type and the mutant NT31. Basal
thermo-tolerance was determined as the percentage of cells surviving
after a direct shift of cultures from 30 °C to a lethal temperature
of 50 °C. Cells were incubated at 50 °C for 20 min in the light
and cultured on a plate at 30 °C for a week, and then the number of
colonies was counted. As shown in Fig.
7A, the survival rate
of NT31 (0.2%) was 2 orders of magnitude lower than that of the wild
type (49%), indicating that the expression of orf7.5
plays an important role in basal thermo-tolerance.
Acquired thermo-tolerance was determined as the percentage of cells
surviving after exposure to 50 °C following a 60-min pretreatment at
42 °C (Fig. 7B). 42 °C is high enough to induce at
least the htpG gene and the groESL operon (data
not shown). The pretreatment gave full protection against the lethal
temperature to the wild type. Although the mutant NT31 also acquired a
remarkable thermo-tolerance through pretreatment, the survival
rate of the mutant was half that of the wild type.
Complementation of NT31 with a DNA Fragment Containing
orf7.5--
The 0.8-kbp DNA fragment containing
orf7.5 (Fig. 1A) was introduced into a
neutral site of the NT31 chromosome, resulting in NI52. Both the growth
of NI52 at 30 and 45 °C under the light intensity of 30 µE/m2/s and the color of the NI52 cultures were
indistinguishable from those of the wild type (Fig.
8B and data not shown). On the
other hand, growth of NT31 ceased after 1 day at 45 °C (Fig.
8B), and the color of the culture became white and
transparent after a few days at that temperature (data not shown). The
DNA fragment contains only the first 16 codons of the
purB-coding region (Fig. 1B). Thus, this result
excludes the possibility that the thermolability of NT31 is due to a
polar effect on the expression of the purB gene (located
downstream of orf7.5) caused by the insertion of an
antibiotic gene in orf7.5.
The Translation of orf7.5 Is Essential for the
Thermo-tolerance--
To test the hypothesis that the Orf7.5
protein is involved in thermo-tolerance, we decided to introduce either
a frameshift or a point mutation within the
orf7.5-coding region and attempted to determine
whether these mutated orf7.5 genes can complement NT31, returning it to the wild type phenotype. Fig. 8A
presents the DNA sequence of the 5'-region of orf7.5
and, below it, the sequences of the two mutated
orf7.5 genes introduced into the neutral sites of the
NI71 and NI81 chromosomes. In both NI71 and NI81, a nonsense mutation
was generated by either inserting or changing a single nucleotide.
Neither mutant could complement NT31, returning it to the wild type
phenotype when mutant growth at 45 °C was compared with the wild
type (Fig. 8C). Thus, the translation product of
orf7.5 appears to be essential for the thermo-tolerance of Synechococcus sp. PCC 7942.
A typical Shine-Dalgarno sequence (AGGAGGA) (26) was found
upstream of the first ATG of orf7.5 (Fig.
1B). In fact, the putative sequence was recognized by
E. coli ribosomes. We subcloned the 2.0-kbp
HindIII-EcoRI fragment (Fig. 1A)
containing the whole sequence of orf7.5 and 131-bp
upstream sequence of its start codon into pBluescript II KS (+). An
E. coli strain harboring the plasmid produced large amounts
of a 7.5-kDa protein when 1 mM
isopropylthio- Possible Regulation of the Expression of the groESL Operon
and htpG Gene by orf7.5--
We attempted to determine whether
the interruption of orf7.5 may have some effect on
expression of other heat shock genes, thus causing the
temperature-sensitive phenotype described above. The accumulation of a
major transcript of 2.3 kb that hybridized with a labeled RNA
complementary to the bicistronic groESL mRNA was
enhanced in the wild type cells by heat shock at 45 °C (Fig. 9). However, it was strongly inhibited in
NT31. An RNA probe complementary to the htpG mRNA
hybridized with a major transcript of 1.5 kb and a minor one of 2.2 kb.
These mRNAs in the wild type cells increased in response to the
heat shock (Fig. 9). The size of the larger mRNA corresponded to
that reported previously for the htpG gene (7). The smaller
mRNA may be a degradation or processed product of the 2.2-kb
htpG mRNA. Independent replicate experiments utilizing
different cultures also resulted in the major transcript of 1.5 kb. The
accumulation of those mRNAs was only slightly inhibited in NT31
(Fig. 9).
This report presents a novel heat shock gene,
orf7.5, cloned from a mesophilic
Synechococcus sp. PCC 7942. The gene,
orf7.5, encodes a putative polypeptide of 63 amino
acids with a predicted molecular mass of 7,455 Da and a pI of 4.97. We
could not find any nucleotide or amino acid sequences in the data bases
(GenBankTM, EMBL, Swissprot or Cyanobase) that
exhibit significant homology to this orf. The reason for the
absence of a homologous sequence in data bases may be that
orf7.5 is a small orf and located within a
larger orf.
Northern blot and primer extension analyses revealed two transcripts of
orf7.5 that have different 5'-ends (Figs. 2 and 3). The results suggest that either orf7.5 has two
transcription start sites or the smaller transcript is a degradation
product of the larger one. The absence of any obvious promoter
sequences preceding P2 suggests that the 330-base mRNA is a
degradation product of the 400-base mRNA. However, we cannot
eliminate the possibility of the presence of an unknown promoter. If
this is the case, the apparent difference in the amount of the two
transcripts transiently accumulated upon heat shock (Figs. 2 and 3) may
be due to the involvement of different promoters for the two transcripts.
Despite being transcribed from an apparent vegetative sigma
factor-dependent promoter sequence, the transcript of
orf7.5 was heat-inducible. Our results suggest that
an unknown regulatory mechanism suppresses the expression of
orf7.5 in cyanobacteria under non-heat shock
conditions. The upstream region of orf7.5 contains no
CIRCE element, indicating that the heat induction mechanism for the
orf7.5 gene must be different from that for the
groESL operon. The regulatory difference between the two
genes is evident in the differential effect of chloramphenicol. The accumulation of groESL mRNA was completely inhibited by
chloramphenicol added before heat shock, whereas that of
orf7.5 mRNA was promoted remarkably (Fig. 4).
Thus, heat-induced de novo synthesis of a protein(s) may be
necessary for heat induction of the groESL operon, whereas
it may repress that of the orf7.5 gene. As shown in
Fig. 9, the inactivation of orf7.5 abolished the
heat-induced accumulation of the groESL mRNA.
Furthermore, it is the translation product of orf7.5
that exerts the thermo-resistance (Fig. 8). An attractive hypothesis is
that the Orf7.5 protein acts as a positive regulator for heat
induction of the groESL operon, whereas it is a negative regulator for the orf7.5 induction. Thus, the
orf7.5 gene may be self-regulated in its transcription.
The photoautotrophic growth of the orf7.5 mutant,
NT31, was strongly inhibited at 45 °C (Fig. 6). The mutant was much
less viable than the wild type at 50 °C (Fig. 7A). The
thermolability phenotype of the mutant indicates that
orf7.5 plays a role in the growth and survival of
Synechococcus sp. PCC 7942 at high temperatures. The ability
of the mutant to gain tolerance to short exposures at 50 °C by
pre-exposure to 42 °C was also reduced (Fig. 7B). The
thermolability phenotype of the mutant is not caused by a polar effect
of the insertion of an antibiotic resistance gene into
orf7.5 on the expression of the downstream gene
purB. This is because a 0.8-kbp fragment containing
orf7.5 could complement NT31 (Fig. 8B).
Furthermore, the same fragment containing orf7.5 with
a nonsense mutation could not complement NT31 back to the wild type
phenotype (Fig. 8C). These results strongly support the idea
that the Orf7.5 protein, but not RNA, is involved in the
thermo-tolerance of Synechococcus sp. PCC 7942. The high
temperature-sensitive phenotype of NT31 may be due to the reduced
expression of the groESL operon (Figs. 9).
Our present results suggest that the novel heat shock protein encoded
by orf7.5 is involved in the heat induction of the
groESL operon and plays an important role in thermal stress
management in cyanobacteria. We postulate that the translation product
of orf7.5 alone or together with other proteins
interacts with the groESL operon at the DNA level and/or RNA
level to regulate their heat induction.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP by the
multiprime-labeling method as directed by the manufacturer (Amersham
Pharmacia Biotech). The probe was used to screen a
Synechococcus sp. PCC 7942 genomic library constructed in
bacteriophage
-DASH vector. Bacteriophage DNA from positive plaques
was prepared by the liquid culture method, and further screening was
performed by Southern blot analysis (18) after digestion of the DNA by
EcoRI. A 6.5-kbp fragment that hybridized with the above
probe was subcloned into pBluescript II KS (+) (Stratagene, La Jolla,
CA). A 5.0-kbp XhoI-EcoRI fragment was generated
by digestion of the 6.5-kbp EcoRI fragment with XhoI and was subcloned into pBluescript II KS (+).
Overlapping deletions of the 5.0-kbp XhoI-EcoRI
fragment were obtained by the Erase-a-Base System (Promega, Madison,
WI). With M13KO7 as a helper phage, single-stranded templates from both
strands were generated for sequencing.
-32P]ATP by T4 polynucleotide kinase
(New England Biolabs, Beverly, MA), purified through a NAP-10 column
(Amersham Pharmacia Biotech), and then used as a primer. Labeled primer
(2 pmol) and 20 µg of total RNA in 30 µl of hybridization buffer
(40 mM Pipes, pH 6.4, 1 mM EDTA, 0.4 M NaCl, and 80% (v/v) formamide) was heated at 85 °C
for 10 min and annealed at 30 °C for 12 h. After ethanol precipitation, the primer and total RNA were dissolved in a final volume of 20 µl of reverse transcriptase buffer (50 mM
Tris-HCl, pH 8.3, 75 mM KCl, 3 mM
MgCl2, 10 mM dithiothreitol, and 0.5 mM dNTPs). Primer extension was carried out by adding 200 units of SuperscriptTM II RNase H
reverse
transcriptase (Life Technologies, Inc.) and incubating at 42 °C for
50 min. Subsequently, 1 µl of 0.5 M EDTA and 1 µl of
RNase A (10 µg/ml) were added and then incubated at 37 °C for 30 min. The sample was extracted with phenol-chloroform, precipitated with
ethanol, and resuspended in 4 µl of 0.5 M EDTA, pH 7.4, and 6 µl of formamide loading buffer (80% (v/v) formamide, 10 mM EDTA, pH 8.0, 1 mg/ml xylene cyanol, and 1 mg/ml
bromphenol blue). After heat denaturation, a 5-µl sample was loaded
onto a 6% polyacrylamide, 7 M urea sequencing gel for
electrophoresis. Products of dideoxynucleotide sequencing reactions
performed with the same primer and the cloned 5.0-kbp
XhoI-EcoRI fragment as a template were run in
parallel to allow determination of the end points of the extension products.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A, restriction map of the
sequenced genomic region containing the orf14.1,
orf7.5, and purB genes from
Synechococcus sp. PCC 7942. Open reading frames are shown by
bold arrows with an arrowhead showing the
direction of each frame. The region of the NcoI fragment
which was used for the preparation of the DNA probes for Southern and
Northern blot analyses is indicated by a shorter dotted
line. The 0.8-kbp fragment that was used for a complementation
test of the orf7.5 mutant and also for the
preparation of the RNA probe for Northern blot analysis is indicated by
a longer dotted line. B, the nucleotide sequence
of the region that contains orf14.1,
orf7.5, and the 5'-end of purB.
Nucleotides are numbered from the 5'-end, and the deduced amino acid
sequences of orf14.1, orf7.5, and
purB are shown below the corresponding DNA sequences. The
deduced amino acid sequence of orf7.5 is
shaded. Putative 10 and
35 sequences are
underlined. The putative Shine-Dalgarno sequence is
double-underlined. Putative transcription start sites (P1
and P2), determined by primer extension analysis, are indicated with
vertical arrows. A sequence of dyad symmetry downstream of
orf7.5, which could act as a transcriptional
terminator, is indicated with arrows.
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Fig. 2.
Northern blot analysis of the
orf7.5 transcripts. Total RNA was
extracted from cells incubated for 0, 15, 30, 45, and 60 min
(lanes 1-5, respectively) after shifting cultures from 30 to 40 °C (A) or 45 °C (B). The
NcoI fragment (shown in Fig. 1A) was labeled with
[ -32P]dCTP by a multiprime-labeling method according
to the manufacturer's specification and used as the probe for Northern
blot analysis. After the analysis of orf7.5, the blot
was stripped off with boiling water containing 0.2% (w/v) SDS 3 times,
10 min each. After being verified to be nonradioactive, the membrane
was reused for reprobing with a radiolabeled 740-bp DNA fragment
containing the entire coding region of the psaD gene of
Synechocystis sp. PCC 6803 (23) (C).
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Fig. 3.
Mapping of the 5'-end of the
orf7.5 mRNA by primer extension
analysis. Primer extension reactions were performed with total RNA
isolated from Synechococcus sp. PCC 7942 cells before
(lane 1) and after shifting from 30 to 45 °C for 15, 30, 60, and 120 min (lanes 2-5, respectively). T,
G, C, and A indicate the
dideoxy-sequencing ladder obtained with the same oligonucleotide as the
primer. The potential transcription start points, P1 and P2, are marked
by asterisks.
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Fig. 4.
The effect of chloramphenicol on the
accumulation of orf7.5 mRNA
(A) or groESL mRNA
(B) in Synechococcus sp.
PCC 7942. Cells were grown at 30 °C (lane 1)
and then divided into two portions. To a 200-ml culture, 2.0 ml of
100% ethanol was added, and the culture was incubated at 30 °C for
5 min (lane 2, 0 min). Then, the culture was shifted to
45 °C for 15, 30 and 60 min (lanes 3-5, respectively)
(shown by closed circles). To another 200-ml culture, 2.0 ml
of chloramphenicol (30 mg/ml in 100% ethanol) was added, and the
culture was incubated at 30 °C for 5 min (lane 6). Then
the culture was shifted to 45 °C for 15, 30, and 60 min (lanes
7-9, respectively) (shown by open circles). Northern
blot analysis with total RNA (10 µg of nucleic acid) obtained at
various times was carried out with the radiolabeled NcoI
fragment, and the combined levels of the 400- and 330-base
orf7.5 mRNAs were quantitated with a Bio-imaging
analyzer (BAS1000 MacBAS, Fuji Photo Film). After the analysis of the
orf7.5 mRNA, the blot was stripped as described
in Fig. 2. The membrane was reprobed with a radiolabeled 540-bp PCR
product containing a part of the groEL1 gene of S. vulcanus (13), and the transcript levels were quantitated as for
the orf7.5 mRNA.
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Fig. 5.
Construction of a clone for insertional
inactivation of orf7.5. The region of
cloned genomic DNA of Synechococcus sp. PCC 7942 in
pBluescript II KS (+) is indicated as a line. Under the
line, orf7.5 and purB are shown
as arrows, with an arrowhead showing the
direction of each open reading frame. In the same way, a
kanamycin-resistant gene cassette (designated as Kmr) is
shown above the line.
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Fig. 6.
Photoautotrophic growth of the wild type
(diamonds) and the mutant strain, NT31
(circles), at 30 and 45 °C. The growth of the
cells was monitored by measuring apparent absorbance of the cultures at
730 nm.
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Fig. 7.
A, the basal thermo-tolerance of the
wild type and the mutant strain NT31. The wild type and the mutant
strain cells grown at 30 °C were shifted directly to 50 °C and
incubated for 20 min in the light. The % survival was calculated as
the percentage of the number of cells surviving after the incubation,
referring to the number of cells at 30 °C as 100%. B,
the acquisition of thermo-tolerance by the wild type and the mutant
strain NT31. The wild type and the mutant strain cells grown at
30 °C were given a pretreatment at 42 °C for 60 min in the light
and then exposed to 50 °C for 20 min in the light. The % survival
was calculated as the percentage of the number of cells surviving after
the incubation, referring to the number of cells surviving after the
pretreatment as 100%.
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Fig. 8.
Complementation of NT31 by the wild type
orf7.5 gene but not by the
orf7.5 gene with nonsense mutations.
A, DNA sequence of the 5'-region of
orf7.5. In NI52, the 0.8-kbp DNA fragment (see Fig.
1A) containing the wild type orf7.5 gene
was introduced into the neutral region of the NT31 chromosome. In the
neutral region of NI71 or NI81, an orf7.5 segment was
introduced in which G was inserted at its ninth codon, or C was changed
to T at its third codon, respectively. The asterisk
indicates a stop codon. B, photoautotrophic growth of the
wild type (diamonds) and the mutant strains, NT31
(circles), and NI52 (triangles, dotted
line) at 45 °C. C, photoautotrophic growth of the
wild type (diamonds) and the mutant strains, NT31
(circles), NI71 (triangles, dotted
line), and NI81 (squares, dotted line) at
45 °C. The growth of the cells was monitored by measuring apparent
absorbance of the cultures at 730 nm.
-D-galactoside was added to the culture
(data not shown). The amino-terminal sequence and the molecular mass of
the recombinant protein revealed that it was the translation product of
orf7.5 (data not shown).
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Fig. 9.
Northern blot analyses of the
orf7.5, groESL, and
htpG transcripts in the wild type and the mutant
strain, NT31. Total RNA was extracted from cells before and after
a temperature shift from 30 to 45 °C for 15 min. Total RNA in the
amount of 2.5 µg was applied to each lane. The
digoxigenin-labeled RNA complementary to either groESL1 from
S. vulcanus, orf7.5, or htpG
from Synechococcus sp. PCC 7942 was used as a probe.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We are grateful to Professor Susan S. Golden for generously providing the vector pAM1573. We thank Professor Alice Schroeder for reading this manuscript and Professor Naoki Sato for conducting homology searches.
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FOOTNOTES |
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* This work was supported in part by Grant-in-aid for Scientific Research (C) 11640641 from the Ministry of Education, Science, Sports, and Culture of Japan to H.N.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number AB002694.
To whom correspondence should be addressed: Tel.: 81-48-858-3403;
Fax: 81-48-858-3384; E-mail: nakamoto@post.saitama-u.ac.jp.
§ Recipient of a Junior Research Associate Fellowship from RIKEN. Current address: National Institute of Agrobiological Resources, Kannondai, Tsukuba, Ibaraki 305-8602, Japan.
Published, JBC Papers in Press, May 7, 2001, DOI 10.1074/jbc.M101717200
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ABBREVIATIONS |
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The abbreviations used are: Hsp, heat shock protein; PCR, polymerase chain reaction; Pipes, 1,4-piperazinediethanesulfonic acid; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; bp, base pair(s); kbp, kilobase pair(s).
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