Fiona J. Woodger

Identification of a SulP-type bicarbonate transporter in marine cyanobacteria

Cyanobacteria possess a highly effective CO2-concentrating mechanism that elevates CO2 concentrations around the primary carboxylase, Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase). This CO2-concentrating mechanism incorporates light-dependent, active uptake systems for CO2 and HCO–3. Through mutant studies in a coastal marine cyanobacterium, Synechococcus sp. strain PCC7002, we identified bicA as a gene that encodes a class of HCO–3 transporter with relatively low transport affinity, but high flux rate. BicA is widely represented in genomes of oceanic cyanobacteria and belongs to a large family of eukaryotic and prokaryotic transporters presently annotated as sulfate transporters or permeases in many bacteria (SulP family). Further gain-of-function experiments in the freshwater cyanobacterium Synechococcus PCC7942 revealed that bicA expression alone is sufficient to confer a Na+-dependent, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document} uptake activity. We identified and characterized three cyanobacterial BicA transporters in this manner, including one from the ecologically important oceanic strain, Synechococcus WH8102. This study presents functional data concerning prokaryotic members of the SulP transporter family and represents a previously uncharacterized transport function for the family. The discovery of BicA has significant implications for understanding the important contribution of oceanic strains of cyanobacteria to global CO2 sequestration processes.

Inorganic Carbon Limitation Induces Transcripts Encoding Components of the CO2-Concentrating Mechanism in Synechococcus sp. PCC7942 through a Redox-Independent Pathway

The cyanobacterial CO2-concentrating mechanism (CCM) allows photosynthesis to proceed in CO2-limited aquatic environments, and its activity is modulated in response to inorganic carbon (Ci) availability. Real-time reverse transcriptase-PCR analysis was used to examine the transcriptional regulation of more than 30 CCM-related genes in Synechococcus sp. strain PCC7942 with an emphasis on genes encoding high-affinity Ci transporters and carboxysome-associated proteins. This approach was also used to test hypotheses about sensing of Ci limitation in cyanobacteria. The transcriptional response of Synechococcus sp. to severe Ci limitation occurs rapidly, being maximal within 30 to 60 min, and three distinct temporal responses were detected: (a) a rapid, transient induction for genes encoding carboxysome-associated proteins (ccmKLMNO, rbcLS, and icfA) and the transcriptional regulator, cmpR; (b) a slow sustained induction of psbAII; and (c) a rapid sustained induction of genes encoding the inducible Ci transporters cmpABCD, sbtA, and ndhF3-D3-chpY. The Ci-responsive transcripts investigated had half-lives of 15 min or less and were equally stable at high and low Ci. Through the use of a range of physiological conditions (light and Ci levels) and inhibitors such as 3-(3,4-dichlorophenyl)-1,1dimethylurea, glycolaldehyde, dithiothreitol, and ethoxyzolamide, we found that no strict correlation exists between expression of genes known to be induced under redox stress, such as psbAII, and the expression of the Ci-responsive CCM genes. We argue that redox stress, such as that which occurs under high-light stress, is unlikely to be a primary signal for sensing of Ci limitation in cyanobacteria. We discuss the data in relation to current theories of CO2 sensing in cyanobacteria.

Sensing of Inorganic Carbon Limitation in Synechococcus PCC7942 Is Correlated with the Size of the Internal Inorganic Carbon Pool and Involves Oxygen

Freshwater cyanobacteria are subjected to large seasonal fluctuations in the availability of nutrients, including inorganic carbon (Ci). We are interested in the regulation of the CO2-concentrating mechanism (CCM) in the model freshwater cyanobacterium Synechococcus sp. strain PCC7942 in response to Ci limitation; however, the nature of Ci sensing is poorly understood. We monitored the expression of high-affinity Ci-transporter genes and the corresponding induction of a high-affinity CCM in Ci-limited wild-type cells and a number of CCM mutants. These genotypes were subjected to a variety of physiological and pharmacological treatments to assess whether Ci sensing might involve monitoring of fluctuations in the size of the internal Ci pool or, alternatively, the activity of the photorespiratory pathway. These modes of Ci sensing are congruent with previous results. We found that induction of a high-affinity CCM correlates most closely with a depletion of the internal Ci pool, but that full induction of this mechanism also requires some unresolved oxygen-dependent process.

The role of GAMYB transcription factors in GA-regulated gene expression

A gibberellin- and abscisic acid-regulated MYB, GAMYB, was first identified as an activator of GA-regulated genes in cereal aleurone. Here we review recent advances made in delineating the signaling events related to GAMYB expression and function in aleurone. In addition, there is a growing body of evidence that GAMYB plays an important role in other aspects of plant growth and development, including anther development, stem elongation, floral initiation and seed development.

Transcriptional Regulation of the CO2-Concentrating Mechanism in a Euryhaline, Coastal Marine Cyanobacterium, Synechococcus sp. Strain PCC 7002: Role of NdhR/CcmR

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.

A Synechococcus PCC7942 _ccmM (Cyanophyceae) mutant pseudoreverts to air growth without regaining carboxysomes

In the cyanobacteria, intracellular structures called carboxysomes function to concentrate CO2 around the relatively inefficient CO2‐fixing enzyme RUBISCO. Mutants devoid of carboxysomes, such as the Synechococcus PCC7942 ΔccmM mutant, are able to grow at high‐CO2 (2% v/v CO2 in air) but perish in air. By growing ΔccmM in air containing 0.23% CO2, and then normal air (0.037%) it was possible to isolate spontaneous pseudorevertant colonies (PsrΔccmM) that still lack ccmM and carboxysomes but can grow in air with a maximal doubling time ∼4‐fold greater than wild type. Inorganic carbon (Ci) uptake and accumulation in PsrΔccmM was >3‐fold greater than wild type and ΔccmM. However, little, or no, change was detected in the transcript abundance of known genes involved in Ci uptake. Significant differences only in the icfA and rbcLS mRNA levels were observed in PsrΔccmM that corresponded with measured changes in IcfA (carboxysomal carbonic anhydrase) and RUBISCO contents. Genomic sequencing spanning icfA, rbcLS, and various Ci transporter and associated gene regions did not identify mutations unique to PsrΔccmM that might impart the growth‐in‐air phenotype. Moreover, the phenotype could not be conferred to ΔccmM by complementation studies with PsrΔccmM genomic DNA fragments, suggesting that it probably results from two or more, as yet unidentified, mutations. The generation of PsrΔccmM demonstrates, for the first time, that carboxysomes are not obligatory for the growth of cyanobacteria in air. We speculate that PsrΔccmM has gained some form of post‐translational up‐regulation of Ci transport.