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Link to original content: http://www.ncbi.nlm.nih.gov/pubmed/17307862
Transcriptional regulation of the CO2-concentrating mechanism in a euryhaline, coastal marine cyanobacterium, Synechococcus sp. Strain PCC 7002: role of NdhR/CcmR - PubMed Skip to main page content
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. 2007 May;189(9):3335-47.
doi: 10.1128/JB.01745-06. Epub 2007 Feb 16.

Transcriptional regulation of the CO2-concentrating mechanism in a euryhaline, coastal marine cyanobacterium, Synechococcus sp. Strain PCC 7002: role of NdhR/CcmR

Fiona J Woodger  1 , Donald A Bryant, G Dean Price
Affiliations

Transcriptional regulation of the CO2-concentrating mechanism in a euryhaline, coastal marine cyanobacterium, Synechococcus sp. Strain PCC 7002: role of NdhR/CcmR

Fiona J Woodger et al. J Bacteriol. 2007 May.

Abstract

Cyanobacterial photosynthesis occurs in radically diverse habitats and utilizes various forms of a CO(2)-concentrating mechanism (CCM) featuring multiple inorganic carbon (C(i)) transporters. Cyanobacteria from dynamic environments can transform CCM activity depending on C(i) availability, and yet the molecular basis for this regulation is unclear, especially in coastal strains. LysR family transcription factors resembling the Calvin cycle regulator CbbR from proteobacteria have been implicated in the expression of C(i) transporter genes in freshwater cyanobacteria. Our survey of related factors revealed a group of divergent CbbR-like sequences confined to freshwater and coastal or offshore cyanobacteria. Inactivation of the single gene (termed ccmR) from this variable cluster in the euryhaline (coastal) strain Synechococcus sp. strain PCC 7002 led to constitutive expression of a high-affinity CCM. Derepression of HCO(3)(-) transporter gene transcription, including that of BicA, a recently discovered HCO(3)(-) transporter (G. D. Price et al., Proc. Natl. Acad. Sci. USA 101:18228-18233, 2004), was observed. A unique CcmR-regulated operon containing bicA plus 9 open reading frames encoding likely Na(+)/H(+) antiporters from the CPA1 and Mnh families was defined that is essential for maximal HCO(3)(-)-dependent oxygen evolution. The promoter region required for C(i)-regulated transcription of this operon was defined. We propose that CcmR (and its associated regulon) represents a specialization for species inhabiting environments subject to fluctuating C(i) concentrations.

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Figures

FIG. 1.
FIG. 1.
Turnover of mRNA encoding Ci transporters in Synechococcus strain PCC 7002 cells. Exponentially growing high-Ci cells were swapped to buffer containing approximately 100 μM Ci and bubbled with CO2-free air for 30 min. At this time (time zero) cells were transferred to either CO2-free air or 2.0% CO2 in the presence or absence of 200 μg of RIF ml−1. The relative abundance of chpY, sbtA, bicA, and porB mRNA was determined by quantitative RT-PCR (n = 4). Symbols represent mRNA abundance relative to the 0-min amount (set at 100%) ± the standard error (SE).
FIG. 2.
FIG. 2.
CbbR-like genes in Synechococcus sp. strain PCC 7002. (A) Phylogenetic tree of proteins with >27% amino acid identity to Rhodobacter sphaeroides CbbR from cyanobacteria representing diverse habitats. Proteins were aligned by using CLUSTAL W and the dendrogram generated in the TREEVIEW program. Species name abbreviations: alr/all, Nostoc sp. strain PCC 7120; Avar, Anabaena variabilis ATCC 29413; Cwat, Crocosphaera watsonii WH8501; gll/glr, Gloeobacter violaceus (strain PCC 7421); Npun, Nostoc punctiforme ATCC 29133; PMM, Prochlorococcus marinus (subsp. pastoris, strain CCMP 1378/MED4); Selo, Synechococcus sp. strain PCC 7942 (elongatus); slr/sll, Synechocystis sp. strain PCC 6803; SynW8102, Synechococcus sp. strain WH8102; 7002, Synechococcus sp. strain PCC 7002; tlr/tll, Thermosynechococcus elongatus strain BP-1; Tery, Trichodesmium erythraeum IMS101; YSA′ and YSB′, Synechococcus sp. strain Yellowstone A-Prime and B-Prime. Locus tags are given for the Anabaena, Synechocystis strain PCC 6803, and Gloeobacter violaceus genes. GOIs from the Integrated Microbial Genomes database (DOE, Joint Genome Institute; http://img.jgi.doe.gov/cgi-bin/pub/main.cgi) are used elsewhere except for the Yellowstone species, for which the GenBank accession numbers are given. (B) Physiological phenotype of the group 2 “ccmR” deletion mutant. Rates of Ci-dependent O2 evolution for wild-type (WT) and ΔccmR cells grown continuously with 2% (vol/vol) CO2 in air (two replicates are shown). (C) Structure of the ccmR genomic region in Synechococcus sp. strain PCC 7002. Possible cotranscribed regions are indicated with arrows (dashed lines represent the range of 5′ and 3′ delimits for each mRNA as indicated by RT-PCR; see Table 5). Predicted stem and loop structures are shown. ndhF3/D3, NAD(P)H-dehydrogenase (NDH-13) subunit genes; chpY/cupA, gene for putative CO2 hydration protein associated with NDH-1; orf133, conserved gene of unknown function associated with NDH-1 genes; cytP450, cytochrome p450 (PFAM00067); DSBA, DSBA thioredoxin (PFAM 03123).
FIG. 3.
FIG. 3.
Gene expression in ΔccmR. The relative abundance of mRNA for ccmR-neighboring genes (A), bicarbonate transporter genes (B), and carboxysomal genes (C and D) in wild-type and ΔccmR cells after transfer from bubbling with 2% (vol/vol) CO2 in air to bubbling with air alone for 60 min was determined. Transcript changes were determined by quantitative RT-PCR (n = 4), and symbols represent transcript expression relative to the wild-type (WT) 0-min amount (set at 100%) ± the SE. Note the break in the y axis in panels A and B. The experiment was independently replicated, and representative data are shown.
FIG. 4.
FIG. 4.
Transcriptional regulation of the ccmR region in Synechococcus strain PCC 7002. (A) Relative mRNA abundance of the ORFs downstream from ccmR in wild-type cells transferred from bubbling with 2% CO2 in air to bubbling with normal air. Transcript changes were determined by quantitative RT-PCR (n = 4), and symbols represent transcript expression ± the SE relative to the housekeeping gene, rnpA (set at 1.0). (B) Relative mRNA abundance of ccmR in wild-type (WT) or ccmR insertional mutant cells after transfer from bubbling with 2% (vol/vol) CO2 in air to bubbling with normal air. Transcript changes were determined by quantitative RT-PCR (n = 4), and symbols represent transcript expression relative to the wild-type high-Ci amount (set at 100%) ± the SE. Note the break in the y axis in panel A. (C) Predicted promoter structure of the ndhF3 upstream region. Consensus LysR binding sites are in boldface, and putative −35 and −10 elements (as predicted by BPROM) are also shown. The most 5′ TSP determined experimentally is marked with an arrow. (D) Luciferase activity directed by the 550-bp ndhF3 upstream region fused to luxAB genes (pF3:P550) and stably integrated into the genome of Synechococcus sp. strain PCC 7002 cells. Cells were grown at high Ci or with air bubbling for 5 h. A positive Ci-responsive control (pbicA:P722F) consisting of a 722-bp fragment from the upstream region of the bicA gene fused to luxAB, is shown (see Fig. 6A). Relative luminescence units are expressed on a chlorophyll a basis as a percentage of the value obtained for high-Ci cells containing the empty vector control ± the standard deviation.
FIG. 5.
FIG. 5.
Structure, regulation, and function of the bicA genomic region in Synechococcus strain PCC 7002. (A) Map of the bicA genomic region. Cotranscribed regions are indicated with arrows (dashed lines represent the range of 5′ and 3′ delimits for each mRNA as determined by RT-PCR; see Table 6). nhaS3, Na+/H+ antiporter (HMM PF00999); mnhC (HMM PF00420), mnhD1/2 (HMM PF00361), and mnhB (HMM PF04039), NADH:ubiquinone oxidoreductase-like subunits from a putative multisubunit Na+/H+ antiporter; T'glycosylase, transglycosylase (PFAM03562); recB, nuclease (COG2251.1); HC, hypothetical conserved protein. (B) Relative mRNA abundance of the ORFs downstream of bicA in wild-type (WT) cells transferred from bubbling with 2% CO2 in air to bubbling with air. Transcript changes were determined by quantitative RT-PCR (n = 4), and symbols represent transcript expression ± the SE relative to the housekeeping gene, rnpA (set at 1.0). (C) Relative mRNA abundance of ORFs in the bicA region in wild-type (WT) or ΔccmR mutant cells bubbled continuously with 2% (vol/vol) CO2 in air. Transcript changes were determined by quantitative RT-PCR (n = 4), and symbols represent transcript expression relative to the wild-type 0-min amount (set at 100%) ± the SE. (D) HCO3−-dependent oxygen evolution in air-grown wild-type cells and cells containing a deletion between the indicated SmaI and XbaI sites (see panel A) of the nhaS3 and mnhD1 genes (Δnha). Cells were analyzed by membrane inlet mass spectrometry at the indicated Ci concentrations in the presence of added CA or the CO2 uptake inhibitor, EZ. (E) Relative photosynthetic affinity for Ci for cells (part D) as measured by the K0.5(Ci) (Ci concentration for half maximal rate). Note the break in the y axis in panel B.
FIG. 6.
FIG. 6.
Analysis of the bicA promoter region in Synechococcus sp. strain PCC 7002. (A) Luciferase activity directed by bicA upstream elements fused to luxAB genes and stably integrated into the genome of Synechococcus sp. strain PCC 7002 cells. Cells were grown at high Ci or with air bubbling for 5 h. Relative luminescence units are expressed on a chlorophyll a basis as a percentage of the value obtained for high-Ci cells containing the empty vector control ± the standard deviation. Asterisks indicate the location of putative LysR-binding motifs. (B) Predicted promoter structure of the bicA upstream region in Synechococcus sp. strain PCC 7002. Putative LysR binding sites are underlined, and putative −35 and −10 elements (as predicted by BPROM at www.softberry.com) are shown in boldface. The TSPs detected by using RLM-RACE are marked with arrowheads; although it is usual to place functional bias on the longest start points relative to the start codon, the shorter products may be genuine alternative TSPs. Note that the first base of the putative −10 box is a G and not the more usual T.
FIG. 7.
FIG. 7.
Putative promoter structure of the sbtA (A), porB (B), and ccmR (C) upstream regions in Synechococcus sp. strain PCC 7002. Putative LysR binding sites are underlined, −35 and −10 elements are in boldface, and predicted TSPs are marked with an arrowhead (as predicted by BPROM).
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