- Research article
- Open Access
Crosstalk regulation among group 2- Sigma factors in Synechocystis PCC6803
© Lemeille et al; licensee BioMed Central Ltd. 2005
- Received: 22 December 2004
- Accepted: 22 April 2005
- Published: 22 April 2005
The cyanobacterium Synechocystis PCC6803 contains one group 1 (sigA) and four group 2 (sigB, sigC, sigD and sigE) sigma factors. The activity of these multiple sigma factors determines the transcriptional program of this bacterium. We wanted to study the role of the group 2 sigma factors in Synechocystis. We have therefore constructed mutants of each of the group 2 sigma factors and investigated their crosstalk.
We used quantitative RT-PCR analysis to measure the relative abundance of the sig mRNAs in the four sigma mutants. Our data indicate that a network of mutual transcriptional regulation links the expression of the sigma genes. Accordingly, an environmental stress acting on only one of the sigma factors will indirectly modify the expression of most of the other sigma factors. This was confirmed by the transcriptional analysis of the sig mRNAs as a function of nitrogen starvation.
Taken together, our observations suggest that the crosstalk regulation between all group 1 and group 2 genes could be important for the adaptation of the bacterium to different environmental and physiological conditions.
Bacterial sigma subunits of RNA polymerase are global regulators of gene expression, conferring specificity to the recognition of promoters by the core enzyme. Two broad families of sigma factors have been identified: the σ70 type, and the σ54 type factors . The σ54 family regulates a variety of genes such as those involved in chemotaxis, synthesis of structural components of flagella and enzymes involved in the response to nitrogen starvation . The σ70 family is subdivided into three groups . Group 1 comprises the primary sigma factors that control the transcription of housekeeping genes, and these sigma factors are therefore essential for cell viability. Groups 2 and 3 include the so-called alternative sigma factors that coordinate the regulation of gene expression in bacteria on a global level. They direct the transcription of a specific genetic program that allows bacteria to cope with particular environmental changes and stress conditions. Group 2 sigma factors are similar in sequence to primary sigma factors and include proteins such as the stationary-phase-specific sigma factor, RpoS . Group 3 sigma factors show less sequence similarity with those of group 1 and include proteins required for the heat shock response  and motility . The inactivation of a gene encoding a particular groups 2 or group 3 sigma factor usually produces growth defects or other phenotypes under specific physiological or environmental conditions. An E. coli rpoS mutant, e.g., has a pleitropic phenotype: it shows a loss of viability in stationary phase and a decreased resistance to some stresses such as the osmotic stress . In Synechocystis PCC6803, inactivation of the sigF gene, encoding a group 3 sigma factor, leads to the loss of motility and pilus formation . In Synechococcus four mutants of rpoD genes show defects in the circadian expression of the psbAI gene, encoding the protein D1 of the photosystem II reaction center .
The unicellular cyanobacterium Synechocystis sp. strain PCC6803 possesses one group 1 sigma factor, sigA (slr0653), four group 3 sigma factors (sll 0687, sll0856, slr1545, slr1564) and four group 2 sigma factors, sigB to sigE (sll0306, sll0184, sll2012, sll1689) . SigE is involved in the response to nitrogen stress  and a contribution of the SigB/SigD factors to the dark/light adaptation has been reported recently . The synthesis of the other alternative sigma factors is also modulated in response to particular stresses [12–14].
In order to assess the role of alternative sigma factors in Synechocystis, we have chosen to study the group 2 sigma factors. We have analyzed the transcription of all members of this family of sig genes as well as the transcription of the group 1 sigma factor in Synechocystis PCC6803 wild type strain and mutants lacking the group 2 sigma genes. Based on our results we suggest that these sigma factors are linked by a network of mutual regulation that could allow them to act in concert in the global transcriptional control of this bacterium.
Results and discussion
Construction and growth of sigma mutants
Expression of the group 2 sigma genes during normal growth
These expression profiles show that the transcript levels of at least three of these sigma factors depend on the cell density (A750 value) of the culture and suggest that they could be important for the global physiology of this bacterium at all stages of growth.
All sigma factors are expressed in many environmental conditions tested, such as iron and sulfur starvations, and heat and osmotic shocks (data not shown). Similar results were previously obtained in Synechococcus elongatus PCC7942  where all sigma genes were found to be active under many growth conditions. Recently Imamura et al.  measured the concentration of all five sigma factors during normal growth of Synechocystis PCC6803. They found the amounts of these proteins to vary between 1 and 10 fmoles/μg of total protein. These data suggest that all five factors are important for the cellular physiology of Synechocystis PCC6803 under standard conditions. This conclusion is somewhat surprising since four of the five sig genes can be mutated, indicating that neither of them is essential for viability under these conditions. One possible way to reconcile these two divergent observations would be to suppose that the function of the different sigma factors is redundant. According to this model, most genes would be transcribed by more than one sigma factor. Indeed, the sigma genes themselves are transcribed from multiple promoters . Furthermore, it is even possible that the different sigma factors recognize the same promoter. This hypothesis is supported by data obtained from in vitro transcription experiments in which all three different sigma factors (RpoD1, RpoD3, RpoD4) could initiate the transcription of the rrnA, cpcB1A1 and P1a promoters of Synechoccocus sp. strain PCC7942 . This specificity crosstalk among sigma factors is also revealed by an in vivo analysis of psbAI promoter activities in S. elongatus where the principal sigma factor, as well as each group 2 sigma factor, all recognize the psbA1 promoter of this bacterium .
Transcription of sig genes in σ mutants
SigE seems to be a particularly important sigma factor because it controls the expression of three other sig genes. Mutation of the sigE gene had the strongest effects among all mutants inactivating sigma genes, affecting particularly the housekeeping gene sigA and sigB. The role of the housekeeping sigma factor, SigA, remains less well defined. Since inactivation of this gene is lethal, we will have to investigate its role using conditional mutants or biochemical methods.
By quantifying the sigma transcripts in different sigma mutants we have shown that the transcription of the sig genes is controlled by a network of mutual connections between the sigmas. Previous studies in related organisms had also shown a mutual transcriptional regulation of sigma factors. In Synechococcus PCC7942, the rpoD1 gene is transcribed by RpoD3 and RpoD4 factors  and SigC factor has a negative effect on SigB expression . In Borrelia burgdorferi, for example, RpoN regulates the expression of rpoS .
Sigma factors transcription under nitrogen starvation
Our analysis is based on measuring the first level of control of sig genes expression. In other systems, post-transcriptional modifications of sigma factors can occur and may not correlate with transcriptional profiles. The recent data of Imamura et al  suggest however that the intracellular concentration of the sigma factors correlates well with the transcriptional control of these sigma factors (with the possible exception of SigB). The same authors demonstrated that a negative effect of SigC on sigB transcription correlates with the reduction of SigB protein levels in this mutant . These data suggest that transcriptional regulation of sig genes is not drastically modified by post-transcriptional control. This hypothesis is actually under investigation in our laboratory.
Culture and growth conditions
Synechocystis sp. strain PCC6803 was obtained from the Pasteur culture collection. Wild-type and mutant strains were grown at 30°C with continuous illumination at approximately 20 μE m-2 s-1, with 3% CO2 in air, in BG11 medium , buffered with 5 mM Hepes-KOH, pH8. Growth was monitored by measuring the optical density at 750 nm (A750).
For nitrogen starvation, BG11 medium lacking the nitrogen source (NaNO3) was buffered with 20 mM N-tris(hydroxymethyl)methyl-2-aminoethanosulfonic acid (TES) buffer, pH 7.5. Strains used in this condition were grown to an A750 = 1, transferred to the nitrogen-depleted medium and incubated for one week. All cyanobacterial strains were grown on BG11 plates containing 1.5% Difco Bacto Agar. When needed, chloramphenicol was added to a concentration of 10 μg/ml. Growth rates of mutants were compared to a Synechocystis strain carrying the same antibiotic resistance cassette inserted into an inessential gene, ureA. We call this strain the wild-type for our experiments.
DNA manipulation and RNA isolation
Molecular techniques were performed according to standard procedures . Synechocystis genomic DNA was prepared according to the method of Tandeau de Marsac et al. . RNA was extracted from pelleted cells, broken by freezing in liquid nitrogen, and using the RNeasy kit (Qiagen) according to the manufacturer's specifications. Chromosomal DNA was removed by treating RNA preparations with 1 μl of DNAse (at 2U/μl) (Ambion) for 1 hour at 37°C. The concentration of RNA was determined spectrophotometrically.
The sigB (sll0306), sigC (sll0184), sigD (sll2012) and sigE (sll1689) genes  were cloned using the TOPO-TA cloning kit (Invitrogene) and the following primers: sigB-1 and sigB-2, sigC-1 and sigC-2, sigD-1 and sigD-2, sigE-1 and sigE-2.
These genes were then subcloned into pBluescript SK- plasmid (Stratagene) between the ApaI and SpeI sites. A chloramphenicol cassette was inserted at the unique site Bgl II in sigC, Sma I in sigB, and BamH I both in sigE and sigD. The cassette was obtained from the pACYC184 plasmid  by PCR amplification. The primers used were cat-1 and cat-2, they add BamH I and SmaI restriction sites at each end of the amplified sequence.
The SK- vector derivatives containing each interrupted gene were used to transform Synechocystis. Chloramphenicol-resistant transformants that were also ampicillin sensitive were selected and subsequently screened for replacement of the wild-type gene allele with the corresponding mutant. Genomic DNA isolated from individual CmR transformants was verified by PCR.
For each reaction, 1 μl of antisens primer mix at 2.5 μM of each of the primers and 200 ng of total RNA were denaturated at 95°C and chilled quickly on ice. A mix consisting of 4 μl of 5x buffer (250 mM Tris-HCl pH 8.3, 375 mM KCl, 15 mM MgCl2, 50 mM DTT), 0.5 μl of RNase Inhibitor (40U/μl), 1 μl of 5 mM dNTP and 1 μl of MMLV reverse transcriptase enzyme (200U/μl) was added in a total volume of 20 μl, followed by one hour of incubation at 37°C. The composition of the primer mix used in each reverse transcription reaction was dependent on the genes analyzed in the quantitative PCR experiments (for example when rpoA and glnN genes were quantified, the reverse transcription reaction used rpoA-RT and glnN-RT primers). The primers used were: rpoA-RT; sigB, C-RT; sigA, D, E-RT; glnN-RT.
List of primers used in this study. All sequences are written from 5' to 3'.
sigA, D, E-RT
Real-time quantitative PCR
PCR conditions were identical for all reactions. The 25 μl-reaction mixture consisted of 1x master mix buffer (Eurogentec), 0.75 μl of SYBR Green I Dye (Eurogentec), and 1 μl of each primer (2 μM). 5 μl aliquots of the diluted reverse transcription reaction were used as template. PCR amplifications (2' at 50°- 10' at 95° – 40 X [15 '' at 95° – 1' at 60°]) were carried out in a Gen Amp 5700 sequence detection system (Applied Biosystems). The forward (F) and reverse (R) primers used in these PCR reactions were designed using the Primer Express software (Applied Biosystems). For each RT-PCR reaction, the efficiency of the DNAse treatment was verified by an identical parallel PCR reaction, but omitting reverse transcription. Only DNA-free RNAs were used in our experiments. All PCR primers anneal to their target at temperatures comprised between 58 and 60°C and amplify 200-pb fragments internal to the coding sequence of the relevant gene. The primers used in the quantitative-PCR experiments are listed in table 1.
Quantitative analysis of the sample
rpoA Ct values for the different growth conditions tested in this study. The reported values represent the average of 6 measurements obtained from two separate RNA preparations.
Culture condition or genetic background
rpoA Ct value per 20 ng total RNA
28.5 ± 1.03
29 ± 0.27
29.44 ± 0.03
Early stationary phase
28.60 ± 0.52
Late stationary phase
28.15 ± 0.04
Wild type strain
30 ± 0.91
30.29 ± 0.25
29.66 ± 1.06
29.76 ± 0.8
29.85 ± 0.69
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