Department of Biology, Texas A&M University, College Station, TX 77843-3258, USA
Correspondence
Susan S. Golden
sgolden{at}tamu.edu
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ABSTRACT |
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Present address: CSIRO Plant Industry, GPO Box 1600, Canberra ACT 2601, Australia.
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INTRODUCTION |
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The psbAI promoter (PpsbAI) is one of the strongest promoters in S. elongatus (Andersson et al., 2000; Liu et al., 1995
) and is often used to study circadian gene expression in this organism (Andersson et al., 2000
; Golden et al., 1997
). The promoter elements of psbAI have been studied using psbAI : : lacZ and psbAI : : luxAB reporter gene fusions (Nair et al., 2001
). The functional elements of the psbAI promoter include a positive element located between 115 and 54 and a basal promoter extending from 54 to +1. The psbAI promoter is not expressed in Escherichia coli (Schaefer & Golden, 1989
). The psbAI gene has a 35 region characteristic of E. coli
70 promoters, but the 10 region contains the atypical sequence TCTCCT (Golden et al., 1986
).
Here we describe a gene that affects psbAI expression levels, but does not affect the circadian timing of psbAI expression. This gene was found during a search for random overexpression mutants with altered circadian expression of psbAI. One isolate exhibited low amplitude, high bioluminescence expression of PpsbAI : : luxAB. Further experiments confirmed that overexpression of the genomic DNA fragment in the sense orientation from PconII caused the mutant phenotype. Experiments reported here showed that some, but not all genes that we routinely survey for circadian expression are affected and that the factor(s) that alters psbAI expression, PsfR, acts upon the basal promoter region of psbAI.
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METHODS |
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Isolation and sequencing of the psfR genomic DNA fragment.
The overexpression library was transferred to the PpsbAI : : luxAB reporter strain AMC149 by conjugation (Andersson et al., 2000). The overexpression library plasmids integrate into the S. elongatus genome by homologous recombination, as shown in Fig. 1
. Exconjugants were screened for altered circadian expression of psbAI. One exconjugant, AMC371, exhibited low amplitude, high bioluminescence expression of psbAI.
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The pAM1509 genomic DNA fragment was sequenced by the cycle sequencing method. The NCBI GenBank BLAST e-mail server (Altschul et al., 1997) was used for DNA and protein database searches.
Construction of psfR overexpression and knockout plasmids.
Diagrams of the psfR overexpression and knockout plasmids are shown in Fig. 2. A 1·6 kb BamHI fragment from pAM1509 was cloned into the BamHI site of the overexpression vector pAM1451 to create pAM1767 and pAM1768.
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A 1·6 kb BamHI fragment from pAM1509 was cloned into the BamHI site of pUC18 (Vieira & Messing, 1982) to create pAM1761. pAM1761 was cut with HincII and BamHI, pAM1451 was cut with BamHI, and both were blunted with Klenow DNA polymerase. The 1·4 kb HincIIBamHI fragment from pAM1761 was cloned into pAM1451 to create pAM1781 and pAM1782.
pAM2992, used to overexpress psfR in the neutral site 1 (NS1) of S. elongatus (Andersson et al., 2000), was constructed as follows. A 4·1 kb Asp718SphI fragment from pAM1509 was cloned into the Asp718SphI site of pIC20H (Marsh et al., 1984
) to create pAM1801. pAM1801 was digested with SmaI and HincII to remove orfXY, but this also removes 1·4 kb of psfR as a HincII fragment. The psfR HincII fragment was isolated and ligated into the SmaIHincII site of pAM1801. Fragment orientation was checked by restriction mapping and a plasmid with the psfR fragment cloned in the proper orientation was isolated (pAM2886). pNN396 (Elledge & Davis, 1989
) was cut with NotI, blunted with T4 DNA polymerase, then cut with Asp718 to release PconII. The PconII fragment was cloned into the EcoRVAsp718 site of pAM2886 to create pAM2985, which contains psfR (minus orfXY) downstream of PconII in the sense orientation. The PconIIpsfR fragment was released from pAM2985 by digestion with HindIII and cloned into the HindIII site of the NS1 vector pAM2314.
A 2·4 kb XbaI fragment from pAM1509 was cloned into the XbaI site of pUC1819RI to create pAM1762. The pUC1819RI cloning vector is similar to the pUC1819H3 vector described previously (Golden & Wiest, 1988). pUC1819RI contains the small ScaIEcoRI fragment from pUC18 ligated to the large ScaIEcoRI fragment of pUC19. pAM165 was digested with BamHI to release the spectinomycin resistance cassette. The spectinomycin resistance cassette was cloned into the BamHI site of pAM1762 to create pAM1788. Three more psfR knockout constructs were made by inserting the spectinomycin resistance cassette into the ClaI (pAM1787), ScaI (pAM1786) or XbaI (pAM1790) sites within the psfR ORF.
Construction of psfR overexpression strains.
psfR overexpression and knockout plasmids were transferred to reporter strains by conjugation (Andersson et al., 2000) or transformation, followed by selection on BG-11 M agar (Bustos & Golden, 1991
) containing the appropriate antibiotics. For each mutant strain created, a minimum of four independently isolated transformants was assayed for bioluminescence. Ectopic psfR fragments were inserted at neutral sites' in the S. elongatus chromosome: loci at which insertions of ectopic DNA fragments can be made without any apparent phenotype (Andersson et al., 2000
). Neutral site vectors contain cloning sites and a selection marker flanked by neutral site sequence. These vectors can replicate in E. coli, but not in S. elongatus. Transformation occurs by homologous recombination at the neutral site. The ectopic DNA and the selection marker are inserted into the chromosome at the neutral site, while the other vector sequences are lost (Golden et al., 1987
). All other psfR overexpression strains were created by insertion of overexpression plasmids at the psfR locus by single crossover homologous recombination, as shown in Fig. 1
.
Bioluminescence assays.
All reporter strains used in this study (except AMC149) are autonomously bioluminescent. In addition to the luxAB reporter, they contain PpsbAI : : luxCDE, which directs the synthesis of the long-chain aldehyde substrate for luciferase in vivo (Andersson et al., 2000). S. elongatus strains were grown on BG-11 M agar (Bustos & Golden, 1991
); for screening by a Packard TopCount luminometer (Andersson et al., 2000
), samples were inoculated onto BG-11 M agar in 96-well microtitre plates. BG-11 M agar was always supplemented with the appropriate antibiotics for selection purposes. Inoculated Petri or microtitre plates were incubated in constant light for 618 h, then incubated in the dark for 12 h to synchronize the clocks of all of the cells on the plates (Katayama et al., 1999
). The initial screen used a turntable device and CCD camera as described previously (Kondo et al., 1994
). In subsequent analyses (all data shown in this paper) strains were assayed for bioluminescence on a Packard TopCount luminometer. The psfR knockout strain used for the experiment shown in Fig. 8
was monitored for bioluminescence on two different microtitre plates in a single TopCount assay. All other strains were assayed in at least two independent assays.
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RESULTS |
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One of the exconjugants exhibited low amplitude, high bioluminescence expression of the psbAI reporter gene (data not shown, but similar to that shown for derivative strains screened using a Packard TopCount luminometer in Fig. 3a and c). To confirm that this phenotype was caused by overexpression of the genomic DNA fragment driven by PconII, the plasmid was recovered by spontaneous loop-out in the absence of antibiotic selection and reintroduced into a wild-type psbAI reporter strain as described elsewhere (Andersson et al., 2000
). Strains cured of the plasmid had wild-type expression of the psbAI reporter gene, whereas strains transformed with the recovered plasmid (pAM1509) had the low amplitude, high bioluminescence phenotype (data not shown).
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Overexpression of psfR affects a subset of genes in S. elongatus
To determine whether psfR overexpression affects genes other than psbAI, we overexpressed psfR in several different S. elongatus reporter strains. The reporter strains were transformed with pAM1767 or pAM1768 to drive the sense or the antisense orientation, respectively, of orfXY and psfR. Transformants were tested for rhythmic expression of bioluminescence using a Packard TopCount luminometer.
Like psbAI, kaiB is a class 1 gene whose expression peaks at subjective dusk (Liu et al., 1995). KaiB is a component of the central circadian oscillator in S. elongatus (Ishiura et al., 1998
). As shown in Fig. 5
(a and b), overexpression of psfR in either orientation had no effect on kaiB expression.
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We also tested the effect of psfR overexpression on two other well characterized class 1 genes: cikA and sigC. The cikA gene encodes a protein that is a member of the extended bacteriophytochrome family and appears to be part of the input pathway to the circadian clock (Schmitz et al., 2000). Overexpression of psfR in a cikA reporter strain (AMC589) reduced the amplitude of oscillation from the cikA promoter, and often reduced cikA expression levels as well (Fig. 5e
), whereas overexpression of the fragment in the antisense orientation had no effect (Fig. 5f
).
The sigC gene encodes a group 2 sigma factor involved in psbAI expression (Nair et al., 2002). If the sigC gene is inactivated, psbAI expression levels increase, the amplitude of the oscillation increases and the period of psbAI expression increases by 2 h. This long-period phenotype is also seen when sigC is overexpressed. This suggests that other sigma factors can recognize psbAI in the absence of sigC, but wild-type sigC expression is required for normal circadian expression of psbAI. Overexpression of psfR in a sigC reporter strain (AMC1042) resulted in reduced expression and a reduction in the amplitude of oscillation from the sigC promoter approximately 85 % of the time. (Fig. 5g
). In approximately 15 % of samples, overexpression of psfR in AMC1042 did not significantly reduce expression from the sigC promoter. This phenotype was seen in two independently isolated exconjugants, each of which usually displayed reduced expression from the sigC promoter when psfR was overexpressed. Therefore, it is unlikely that this phenotype is the result of suppressor mutations. Overexpression of psfR in the antisense orientation did not affect expression from the sigC promoter (Fig. 5h
).
The psbAI gene is part of a family of three genes that encode the D1 protein in S. elongatus (Golden et al., 1986). Overexpression of psfR in bioluminescent reporter strains for psbAII (AMC1264) and psbAIII (AMC537) resulted in elevated expression from those promoters as was seen for psbAI (Fig. 5i, k
), although no effect was seen when the antisense construct was overexpressed (Fig. 5j, l
). Thus, the psbA family as a whole is responsive to PsfR.
Overexpression of the psfR ORF alone is sufficient for altered psbAI gene expression
To determine whether the small ORFs orfX and orfY are required for elevated psbAI expression, we transformed the psbAI reporter strain AMC412 with plasmid vectors that contain PconIIpsfR but lack orfXY (pAM1781, pAM1769). pAM1781 contains 85 bp upstream of psfR, including 30 bp of orfY. pAM1769 contains 35 bp upstream of psfR (Fig. 2). When S. elongatus is transformed with either plasmid, the plasmid is inserted into the S. elongatus chromosome at the psfR locus by homologous recombination and the entire psfR ORF is driven by PconII (Fig. 1
). As shown in Fig. 6
(a and c), overexpression of psfR from either plasmid was sufficient to elevate psbAI expression and overexpression of these psfR fragments in the antisense orientation had no effect (Fig. 6b and d
). PconIIpsfR was also sufficient to elevate expression from psbAII and psbAIII reporter genes (data not shown).
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psfR acts directly or indirectly at the basal promoter region of psbAI
The psbAI reporter strain AMC412 has end points extending from approximately 75 to +180 relative to the psbAI transcription start site. To determine which elements in the psbAI promoter region are acted upon by psfR (directly or indirectly), we overexpressed psfR in psbAI reporter strains that contain different segments of the psbAI promoter region driving luxAB. AMC776, AMC781 and AMC777 contain sequences extending from 115 to +43, 115 to +1 and 54 to +43, respectively. All three reporter fusions contain the psbAI basal promoter region, which extends from 54 to +1 (Nair et al., 2001). AMC777 lacks the positive regulatory element that extends from 115 to 54 (Nair et al., 2001
), and expression of PpsbAI : : luxAB is much lower in this strain than in AMC776 and AMC781. Note that the AMC777 data presented in Fig. 9
are graphed on a lower scale than the data from AMC776 and AMC781.
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DISCUSSION |
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PsfR overexpression resulted in decreased expression of sigC in many assays. Loss of sigC is known to increase expression from psbAI (Nair et al., 2002). However, the phenotypes are different, as sigC inactivation increases the amplitude of psbAI expression, whereas PsfR overexpression decreases it. Thus, the effect of PsfR overexpression cannot be explained entirely by a loss of sigC. In addition, the psbAI elevation phenotype showed complete penetration, whereas sigC suppression did not.
As shown in Fig. 4, sequence analysis of the putative PsfR protein predicted an N-terminal pseudo-receiver domain that lacks the conserved aspartyl residue that would be needed for phosphoryl transfer from a histidine protein kinase in a bona fide receiver, a C-terminal typical receiver domain and a C-terminal GGDEF domain. Conserved domain searches did not detect a putative DNA-binding domain. While this does not rule out the possibility that PsfR is a DNA-binding protein, the presence of a pseudo-receiver domain suggests that PsfR may regulate psbAI expression via proteinprotein interactions rather than by direct interaction with the psbAI promoter DNA. Although the genomes of other cyanobacteria encode proteins with these motifs, there is no clear homologue of psfR in the available sequences.
A pseudo-receiver domain of known function is present in the AmiR regulatory protein of Pseudomonas aeruginosa (O'Hara et al., 1999). Free AmiR activates expression from the aliphatic amidase operon via a transcription antitermination mechanism. When aliphatic amides are not present, the AmiC protein binds to the pseudo-receiver domain of AmiR, sequestering AmiR and allowing transcription termination to occur, so that the aliphatic amidase operon is not expressed. In a similar fashion, PsfR could indirectly regulate psbAI expression by interacting with a protein that binds to the basal promoter region of psbAI, affecting activity of that protein. Pseudo-receiver domains have been found in the S. elongatus proteins CikA (Schmitz et al., 2000
) and KaiA (Williams et al., 2002
). Members of the Arabidopsis thaliana family of pseudo-response regulators (APRR family), which includes the putative plant clock protein (TOC1), contain pseudo-receiver domains as well (Imamura et al., 1999
; Makino et al., 2000
; Strayer et al., 2000
). All lack the aspartyl residue that would be necessary for two-component system receiver function.
The two receiver-like domains of PsfR may act together to regulate the activity of the protein. Phosphorylation of the C-terminal receiver could make it easier for the N-terminal pseudo-receiver to bind to its protein target, or binding of the pseudo-receiver to its target could regulate phosphorylation of the receiver. In either case, a conformational change in the PsfR protein would result in a change in PsfR activity.
GGDEF domains are found in many multidomain signal transduction proteins; in most, the role of this domain has not been determined. GGDEF domains have sequence similarity to the eukaryotic adenylyl cyclase catalytic domain, suggesting that GGDEF domains could be regulatory enzymes involved in nucleotide cyclization (Pei & Grishin, 2001). Studies of cellulose production in Rhizobium leguminosarum bv. trifolii and Agrobacterium tumefaciens suggest that GGDEF domains are involved in the synthesis of bis-(2',5')-cyclic diguanylic acid (cyclic di-GMP) and that cyclic di-GMP is an activator of cellulose production in these bacteria (Ausmees et al., 2001
). Therefore, the role of GGDEF domains in some proteins is the production of cyclic di-GMP, a signalling molecule in some regulatory pathways. If the GGDEF domain of PsfR produces cyclic di-GMP, perhaps its activity is controlled by conformational changes in PsfR, through phosphorylation of the receiver or proteinprotein interactions with the pseudo-receiver.
Because a phenotype was detected in an overexpression mutant, but not an inactivation mutant, PsfR may be part of a family that serves similar roles in the cell, such that its loss is compensated. It is also possible that the phenotype results from a crosstalk phenotype that does not reflect the true function of PsfR. However, we now know that the conII promoter is low to moderate in strength in S. elongatus, such that overexpression of the protein is likely to be modest (Katayama et al., 1999).
In conclusion, we identified a regulatory gene that affects the expression of a subset of genes in S. elongatus, but does not regulate the S. elongatus circadian clock. Our work shows that overexpression of the psfR ORF is sufficient for elevated psbAI expression and that PsfR acts (either directly or indirectly) at the basal promoter region of psbAI.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 14 November 2003;
revised 7 January 2004;
accepted 12 January 2004.
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