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Journal of Bacteriology, February 2001, p. 1090-1095, Vol. 183, No. 3
Instituto de Bioquímica Vegetal y
Fotosíntesis, Consejo Superior de Investigaciones
Científicas
Received 10 July 2000/Accepted 31 October 2000
The expression of sll1689, an open reading frame from
the cyanobacterium Synechocystis sp. strain PCC 6803 putatively encoding a member of the Eubacterial RNA polymerase consists
of a core enzyme and a sigma factor that directs the complex to a
specific class of promoter sequences. Most eubacterial genomes encode a
number of different sigma factors that recognize different subsets or
groups of promoters. This variety of sigma factors allows basal
expression of certain genes as well as regulated expression of other
genes in response to environmental or developmental signals
(44). Two families of sigma factors have been defined on
the basis of sequence similarity, the Cyanobacteria are a widely distributed group of phototrophic bacteria
that carry out oxygenic photosynthesis and are considered the
precursors of chloroplasts. In cyanobacteria and chloroplasts, the core
of RNA polymerase consists of five subunits ( Cyanobacteria are able to use a number of different nitrogen sources
(17). The regulation of the use of these sources is mediated by the nitrogen control, CAP (catabolite activator
protein)-family transcription factor NtcA. In the absence of ammonium,
NtcA activates the expression of genes required for the assimilation of
nitrogen sources alternative to ammonium (42). A consensus
sequence needed for NtcA binding to DNA (GTAN8TAC) has been
defined (31). The NtcA-activated promoters bear this
sequence, located at about position The complete sequence of the chromosome of Synechocystis sp.
strain PCC 6803 is available (29)
(http://www.kazusa.or.jp /cyano/cyano.html). In the context of our
attempts to identify genes controlled by the global nitrogen regulator
NtcA, we have carried out a search for putative NtcA-activated
promoters in the genomic sequence of strain PCC 6803. In this report,
the identification of sll1689, encoding a protein homologous
to Search for putative NtcA-activated promoters.
A computer
search for sequences of the genome of Synechocystis sp.
strain PCC 6803 that could correspond to putative NtcA-activated promoters was carried out using overlapping 30-kb segments of the
genome sequence and MatInspector software (34). The
consensus sequence needed for NtcA binding, GTAN8TAC, was
found in a total of 367 positions, 44 of which were followed, at a
distance of 20 to 23 nucleotides, by a sequence matching the consensus
for a putative Regulation of expression of rpoD2-V.
In order to analyze
whether the expression of rpoD2-V was in fact
under nitrogen control, Northern hybridization analysis was carried out
with RNA isolated from Synechocystis sp. strain PCC 6803 cells that had been subjected to nitrogen starvation. Standard
molecular biology procedures were performed by published methods
(4, 37). Synechocystis cells were grown in
BG110 medium (medium BG11 [36] without NaNO3)
supplemented with 0.84 g of NaHCO3
liter
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.3.1090-1095.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Nitrogen-Regulated Group 2 Sigma Factor from
Synechocystis sp. Strain PCC 6803 Involved in Survival under
Nitrogen Stress
Universidad de Sevilla, E-41092 Seville, Spain
ABSTRACT
Top
Abstract
Text
References
70 family of sigma
factors, appears to be regulated by the nitrogen control transcription
factor NtcA. Disruption of sll1689 had no noticeable effect
on exponential growth, identifying its product as a member of the group
2, nonessential class of 70-like sigma factors; however,
this disruption decreased the viability of the cells after long periods
of nitrogen starvation. We have named this gene
rpoD2-V. The expression of glnN,
encoding a type III glutamine synthetase, was impaired in strains
bearing an inactivated copy of the rpoD2-V gene.
TEXT
Top
Abstract
Text
References
70 and the
54 families, named after the primary sigma factor and
the factor involved in nitrogen regulation in Escherichia
coli, respectively. All eubacteria contain one or more sigma
factors belonging to the 70 family (44).
Sigma factors that are responsible for the transcription of most genes
in exponentially growing cells and that are essential for survival
constitute group 1 or primary sigma factors; group 2 includes
"primary-like" sigma factors that are nonessential for exponential
cell growth (secondary sigma factors); and group 3 includes the
so-called alternative sigma factors (30). A single group 2 sigma factor, stationary-phase 38, has been found in
E. coli, but multiple group 2 sigma factors are present in
high-GC-content gram-positive bacteria (e.g., Streptomyces spp.), cyanobacteria, and Chloroflexus aurantiacus
(26, 44).
2
'
in cyanobacteria and 2'" in chloroplasts)
(28, 39) and thus is slightly different from that in other
eubacteria (2' tetramer) (9). The
' subunit of eubacterial RNA polymerase is considered to be split
into two subunits in cyanobacteria and chloroplasts, ' and
'", respectively (5). Multiple sigma factors of the 70 family (groups 1 and 2) have been found in
cyanobacteria of the genera Anabaena (7, 8),
Nostoc (11), Synechococcus
(12, 13, 27, 40, 41), Microcystis (2,
3) and Synechocystis (29). The
chromosome of Synechocystis sp. strain PCC 6803 contains five open reading frames (ORFs) that would correspond to sequences encoding primary or primary-like sigma factors (groups 1 and 2) and
three ORFs that would correspond to sequences encoding alternative sigma factors (29). The sequences of the genome of
Anabaena sp. strain PCC 7120 available to date
(http://www.kazusa.or.jp /cyano/anabaena) contain at least eight
ORFs similar to sequences encoding primary or primary-like sigma
factors. No sigma factor belonging to the 54 family has
yet been identified for cyanobacteria. Phylogenetic analysis of
cyanobacterial sigma factor sequences known to date indicates that
group 1 sigma factors are tightly clustered (cluster I) and that group
2 sigma factors form a separate, coherent clade (25, 26)
which appears to be further divided into four clusters, II to V
(25). Each cyanobacterial strain analyzed to date bears members belonging to each group 2 cluster (25).
40.5, and a 10 sequence of the
form TAN3T (18, 31). The ntcA gene
is widely distributed in cyanobacteria (21). In all
strains analyzed to date, the sequence of the NtcA protein is highly
conserved, as is the sequence of the promoters activated by NtcA in
those strains. Insertional mutants of ntcA have been obtained for Synechococcus sp. strain PCC 7942 and
Anabaena sp. strain PCC 7120. However, attempts to isolate a
completely segregated ntcA mutant of
Synechocystis sp. strain PCC 6803 have been unsuccessful (23).
70-like sigma factors, as a nitrogen-regulated gene
as well as the effects of disruption of sll1689 are described.
10 box, TAN3T. Only in 31 of those cases
was the NtcA box located less than 2 kb upstream from an ORF, that is,
in a position that might be considered compatible with a role in
transcription activation. Although some of the identified sequences
were located upstream from ORFs encoding hypothetical proteins of
unknown function, 22 of them were located upstream from either known
genes or ORFs whose predicted products were similar to some proteins in
the databases. Among those, as expected, we could identify the
NtcA-regulated promoter of the glnA gene, encoding glutamine
synthetase (35), and the region upstream from
nirA, encoding nitrite reductase, a gene whose expression
has been shown to be NtcA dependent in Synechococcus sp.
strain PCC 7942 (31) and Anabaena sp. strain PCC 7120 (20). A sequence,
GTAN8TACN21TAN3T, matching the
structure of NtcA-regulated promoters was found 270 nucleotides
upstream from sll1689, one of the five ORFs identified in
the genome of strain PCC 6803 as rpoD-like genes encoding
members of the 70 family of sigma factors
(29). We refer to this ORF as
rpoD2-V because its product belongs to cluster V
of the phylogenetic tree of cyanobacterial primary or primary-like
sigma factors (25).
1 (BG110C medium) and 15 mM
NH4Cl and bubbled with a mixture of CO2 (1%
[vol/vol]) and air. Twice as much
N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES)-NaOH buffer (pH 7.5) as NH4Cl was added
to all NH4Cl-containing media. For RNA isolation, cells
growing exponentially in NH4Cl-containing BG110C medium were harvested at room temperature and either
used directly or washed with and resuspended in BG110C
medium (nitrogen free) and further incubated under culture conditions
for various times. RNA was isolated as previously described
(22). Northern hybridization was carried out using Hybond
N+ membranes according to manufacturer recommendations.
Northern blot experiments performed using the insert of pCSAM82 (a
plasmid containing sll1689 and flanking sequences; see
below) as a probe indicated that the expression of
rpoD2-V was indeed induced in cells subjected to
nitrogen deprivation (Fig. 1). As has
also been observed for the sigC transcript of
Synechococcus sp. strain PCC 7002 (13), the
transcript hybridizing to the rpoD2-V probe appeared as a smear. Control hybridization experiments with different probes showed that the RNA in the filters was not degraded (data not
shown).
View larger version (89K):
[in a new window]
FIG. 1.
Northern blot analysis of the expression of
rpoD2-V in Synechocystis sp. strain
PCC 6803. RNA was isolated from ammonium-grown cells (lane 0) or from
ammonium-grown cells incubated in nitrogen-free medium for 2, 3, 4, 6, or 8 h. Hybridization to a probe for rpoD2-V
was carried out as described in the text. Samples contained 40 µg of
RNA. Sizes of standards (in kilobases) are indicated on the right.
Arrowhead indicates transcripts of about 1.5 kb.
89 to 107 with respect to the putative translational start site of
rpoD2-V) (Fig. 2).
In addition to a constitutive putative tsp located at position 202
with respect to the translational start site of
rpoD2-V, an inducible transcript which
corresponded to a putative tsp located at position 264 was observed.
The sequences located upstream from the inducible tsp corresponded to
the consensus NtcA-activated promoter sequence
(GTAN8TACN21TAN3TN5
tsp) identified in the computer search described above. The
identification of these two putative tsp was confirmed by using
oligonucleotides RD4 (5'-AGA GCA TCA TCC AGA TAG ACC-3',
complementary to positions 103 to 123 with respect to the
putative translational start site of rpoD2-V) and
RD6 (5'-TCA GCG AGG CCA TCC AAA GCC-3', complementary to
positions +77 to +57 with respect to the putative translational start
site of rpoD2-V) (data not shown). The apparent
inconsistency between the Northern and primer extension results could
be due to different stabilities of the constitutive and inducible
transcripts. In any case, the tsp detected at 264 is the one which
reproduces the regulatory pattern observed by Northern analysis for the
rpoD2-V gene.
|
545 to 526 with respect to the
putative translational start site of rpoD2-V) and
RD2 (5'-CCA CTT TCA GCT ATG CGC ACT GCG G-3', complementary to positions 144 to 168 with respect to the putative translational start site of rpoD2-V) and plasmid pCSAM82 (see
below) as a template. Binding assays with purified NtcA protein were
carried out as described previously (33). The results
shown in Fig. 3 indicate that NtcA
specifically bound to the fragment containing the putative inducible
promoter of rpoD2-V. The binding could be
competed by the addition of the same, unlabeled fragment or a fragment
containing the NtcA-regulated promoter of the glnA gene from
Anabaena sp. strain PCC 7120 (19; see the fragment used in
reference 33).
|
255 (13), could in fact correspond to an
NtcA-regulated promoter, since it exhibits a sequence
(GTAN8AAC) that resembles that of the NtcA box,
separated by 22 nucleotides from a putative 10 box of the form
TAN3T.
Disruption of rpoD2-V. A 2-kb DNA fragment comprising rpoD2-V and flanking sequences was amplified by PCR using oligonucleotides RD1 (see above) and RD3 (5'-GAT GCG AGC GAA GAT TTC TG-3', complementary to positions +342 to +323 with respect to the putative translational stop site of rpoD2-V) and total DNA (isolated as described in reference 10) from strain PCC 6803 as a template. The PCR product was cloned in vector pGEM-T (Promega), generating plasmid pCSAM82. This plasmid was digested with BamHI and BglII (both sites are internal to the rpoD2-V gene), and the 1.3-kb Kmr gene cassette C.K1 excised from pRL161 (S.A1/L.HEH1/C.K1; nomenclature as in reference 16) with BamHI was inserted between the BamHI and BglII sites of rpoD2-V, rendering plasmid pCSAM89 (a or b, depending on the orientation of C.K1 with respect to the rpoD2-V gene).
After transformation of Synechocystis sp. strain PCC 6803 with plasmids pCSAM89a and pCSAM89b (14), Kmr transformants were selected and maintained on solid BG110C medium supplemented with 4 mM NH4Cl and 50 µg of kanamycin ml1. To test whether the resulting mutant
strains were homozygous for the mutant chromosomes, PCR amplification
with primers RD1 and RD3 and genomic DNA from the mutants as templates
and Southern hybridization analysis were carried out. Clones homozygous
for the mutated chromosomes were chosen and named CSAM4
(rpoD2-V::C.K1, which carries the gene
cassette in the same orientation as rpoD2-V) and
CSAM5 (rpoD2-V::C.K1, which carries the
gene cassette in the orientation opposite that in
rpoD2-V).
No noticeable effect on cell growth was detected in the mutant strains
with respect to the wild-type strain in nitrate- or ammonium-containing
media. The growth rate constant was determined for single cultures of
the wild-type and mutant strains in 4 mM NH4Cl-containing
medium, and values of 0.62 day1 (wild type), 0.57 day1 (CSAM4), and 0.64 day1 (CSAM5)
were obtained. Thus, rpoD2-V is not essential for
cell viability under nutrient-replete conditions and can be classified as a group 2 sigma factor gene.
Since the expression of rpoD2-V increased under
nitrogen-limiting conditions, a number of different phenotypes related
to the assimilation of nitrogen sources in the mutant strains were tested. The induction of nitrate reductase and glutamine synthetase activities (measured as described previously [19]) took
place in a similar fashion in the wild type and in the mutants (data not shown). Also, the decrease in phycobiliproteins that takes place in
response to nitrogen deprivation (1, 15) was not altered
in the mutants. The effect of rpoD2-V
inactivation on the induction of nitrogen-regulated genes was also
tested by Northern hybridization (Fig.
4). The genes whose expression was
analyzed were amt1 (ammonium/methylammonium permease)
(32), glnB (PII signaling protein)
(22), and glnN (type III glutamine synthetase) (35). The probes used for amt1,
glnB, and glnN were those indicated in the
corresponding references. Quantification of the signals in the Northern
blots shown in Fig. 4, normalized to the signal for the rnpB
probe (43), used as a loading and transfer control, indicated that the expression of amt1 or glnB was
not significantly altered in the rpoD2-V mutants.
Activation of the expression of amt1 or glnB upon
nitrogen deprivation took place earlier than the expression of the
rpoD2-V gene itself (Fig. 1 and 4), making the
participation of RpoD2-V in their expression unlikely. However, activation of the expression of glnN, which under our
experimental conditions took place later than induction of the
expression of amt1 and glnB in response to
nitrogen starvation, was impaired in the mutants; a decrease of about
30% was observed after 4 h of nitrogen starvation (Fig. 4).
Interestingly, glnN recently has been shown to influence the
recovery of Synechococcus sp. strain PCC 7942 cells from
long periods of nitrogen starvation (38).
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1, and further incubated under culture conditions. For
the determination of CFU, appropriate dilutions of liquid cultures were
plated as soft-agar overlays on solid BG110C medium
supplemented with 4 mM NH4Cl. The viability of mutant cells
after long periods of nitrogen starvation was severely altered with
respect to that of wild-type cells. The survival of wild-type cells
subjected to 25 days of nitrogen starvation was about 0.15, a value
similar to that described for Synechococcus sp. strain PCC
7942 (24); however, survival dropped to 7.3 × 104 and 9.1 × 104 for mutant strains
CSAM4 and CSAM5, respectively. Although differences were more dramatic
after this long period of starvation, some differences in survival
between the wild-type and mutant strains could be observed as soon as
after 2 days of nitrogen deficiency.
In order to test whether the lower survival of the mutant strains was
specific for nitrogen stress, we tested the survival of the cells after
starvation for phosphorus, carbon, or sulfur. For this experiment,
cells grown in 15 mM NH4Cl-containing medium were washed
and resuspended in the corresponding nutrient-deficient medium (10 mM
NH4Cl-containing BG110C medium lacking
phosphorus in the case of phosphorus starvation, 10 mM
NH4Cl-containing BG110C medium lacking sulfur
in the case of sulfur starvation, and 4 mM NH4Cl-containing
BG110 medium in the case of carbon starvation). All
cultures except for the carbon-starved ones were bubbled with a mixture
of CO2 (1% [vol/vol]) and air. N2 was
bubbled through the carbon-starved cultures. The survival of wild-type
PCC 6803 cells under phosphorus and carbon stress was similar to that
under nitrogen stress, i.e., about 0.25 after 10 days of starvation. However, whereas the survival of rpoD2-V mutant
cells after nitrogen starvation was decreased with respect to that of
wild-type cells (0.01 after 10 days of nitrogen starvation), the mutant
cells behaved like the wild-type cells under phosphorus and carbon
stress. Survival under sulfur stress was much lower for both the wild type and the mutants, about 0.1 after 2 days of starvation. These experiments indicate that the sigma factor encoded by
rpoD2-V is specifically involved in survival
under severe nitrogen stress.
Concluding remarks. Functions for cyanobacterial nonessential sigma factors have been assigned or suggested only in a few cases. The expression of sigB and sigC of Anabaena sp. strain PCC 7120 was found to be increased in the absence of nitrogen (8). However, single- and double-mutant strains bearing inactivated sigB and/or sigC genes were able to grow fixing nitrogen (8). The expression of sigB and sigC of Synechococcus sp. strain PCC 7002 is also induced under nutrient-limiting conditions (13), and sigE of this strain seems to be implicated in transcription in the stationary phase of growth (27). Mutations in rpoD2 of Synechococcus sp. strain PCC 7942 affect the circadian rhythm of this organism (41), whereas mutations in sigH of Nostoc punctiforme affect the symbiosis of this cyanobacterium with a plant host (11). On the other hand, mutations in sigF of Synechocystis sp. strain PCC 6803, the only cyanobacterial alternative (group 3) sigma factor gene that has been characterized so far, result in a pleiotropic phenotype that includes alterations in pilus formation and motility (6).
The low survival of the Synechocystis sp. strain PCC 6803 rpoD2-V mutants after nitrogen stress could have been due to decreased glnN expression. Unfortunately, we have been unable to analyze the expression of glnN after long periods of nitrogen starvation because of the poor quality of mRNA samples isolated from the starved cells. RpoD2-V could also be involved in the expression of other genes required for survival under conditions of severe nitrogen deficiency. The fact that NtcA-activated promoters bear a10 box of the form TAN3T suggests that NtcA
activates transcription mediated by RNA polymerase containing a
70-like sigma factor. Therefore, NtcA-dependent
rpoD2-V expression might have the effect of
increasing the levels of an appropriate, 70-like sigma
factor to assist NtcA-activated gene expression under conditions of
severe nitrogen starvation.
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ACKNOWLEDGMENTS |
|---|
We thank Enrique Martínez-Force for help with the computer search, Ana Valladares for help in some primer extension experiments, and F. J. Florencio and J. C. Reyes for providing several DNA probes.
This work was supported by grants PB97-1137 and PB98-0481 from Ministerio de Ciencia y Tecnología (Madrid, Spain). A.M.M.-P. was the recipient of postdoctoral contracts from MEC and CSIC (Madrid, Spain) and a fellowship from the Universidad de Sevilla.
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FOOTNOTES |
|---|
* Corresponding author. Mailing address: Instituto de Bioquímica Vegetal y Fotosíntesis, Centro de Investigaciones Científicas Isla de la Cartuja, Avda. Américo Vespucio s/n, E-41092 Seville, Spain. Phone: 34-95-4489523. Fax: 34-95-4460065. E-mail: alicia{at}cica.es.
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