Sensing of inorganic carbon limitation in Synechococcus PCC7942 is correlated with the size of the internal inorganic carbon pool and involves oxygen

Abstract

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.

Figure 1.

Rates of O₂ evolution (A) in wild-type (WT) and ΔchpXΔchpY (ΔXΔY) cells bubbled continuously with 5% or 1% CO₂ in air, and the relative abundance (B) of cmpA and sbtA transcript pools in the same genotypes after transfer from bubbling with 5% to 1% CO₂ in air. Transcript changes were determined by real-time RT-PCR (n = 4), and symbols represent transcript expression relative to the wild-type 0-min amount (set at 100%) ± se. Note the break in the y axis in B. Each experiment was independently replicated and representative data are shown.

Figure 2.

Relative abundance of cmpA (A), sbtA (B), and chpY (C) transcript pools in wild-type (WT) and ΔccmM cells transferred from aeration with 5% CO₂ in air to air bubbling for 4 h. Transcript changes were determined by real-time RT-PCR (n = 4), and symbols represent transcript expression relative to the wild-type 0-min amount (set at 100%) ± se. Note the break in the y axis in A. The experiment was independently replicated and representative data are shown.

Figure 3.

Relative abundance of cmpA, sbtA, and chpY transcript pools in wild-type cells transferred for 30 min from aeration with 2% CO₂ in air to 0.015% ppm CO₂ containing various O₂ concentrations. Transcript changes were determined by real-time RT-PCR (n = 4), and symbols represent the extent of induction after the shift to low Ci as a percentage of the maximum response (set at 100%) ± se. The maximum response (100%) equates to an approximately 800-fold, 250-fold, or 12-fold change for cmpA, sbtA, and chpY, respectively. The experiment was independently replicated and representative data are shown.

Figure 4.

Relative abundance of cmpA (A), sbtA (B), and chpY (C) transcript pools in wild-type cells transferred from aeration with 2% CO₂ in air to 0.015% ppm CO₂ containing 2% or 21% O₂ over 180 min. Transcript changes were determined by real-time RT-PCR (n = 4), and symbols represent the extent of induction after the shift to low Ci at each time point as a percentage of the maximum response (set at 100%) ± se. The maximum response (100%) equates to approximately a 1,300-fold, 350-fold, or 21-fold change for cmpA, sbtA, and chpY, respectively. The experiment was independently replicated and representative data are shown.

Figure 5.

Changes in photosynthetic affinity for Ci, as K_0.5(Ci), in wild-type cells grown with 2% CO₂ in air and switched to aeration with 0.015% CO₂ containing either 2% or 21% O₂ for 1.5, 3.0, 4.5, and 24 h. Symbols represent the average K_0.5(Ci) ± se after the shift to low Ci at each time point (n ≥ 3). Note the break in the x axis.

Figure 6.

Relative abundance of cmpA and sbtA (A) and chpY (B) transcript pools in wild-type (WT) and ΔccmM cells transferred from aeration with 5% CO₂ in air to air bubbling for 30 min in the presence of 300 μm EZ or 10 mm GLY, or with both inhibitors combined. Transcript changes were determined by real-time RT-PCR (n = 4), and bars represent the extent of induction after the shift to low Ci at each time point as a percentage of the wild type + EZ response (set at 100%) ± se. The maximum response (100%) equates to approximately a 1,000-fold, 600-fold, or 14-fold change for cmpA, sbtA, and chpY, respectively. The experiment was independently replicated and representative data are shown.

Figure 7.

Relative abundance of cmpA and sbtA transcript pools in wild-type (WT) and ΔchpXΔchpY cells transferred from aeration with 5% CO₂ in air to bubbling with 0.1% CO₂ containing either 1% or 21% O₂ for 30 min. Transcript abundance was determined by real-time RT-PCR (n = 4). Bars represent the extent of induction after the shift to low Ci as a percentage of the maximum response (set at 100%) ± se. The maximum response (100%) equates to approximately a 350-fold or 100-fold change for cmpA and sbtA, respectively. The experiment was independently replicated and representative data are shown.

Figure 8.

Effect of various concentrations of SHAM on maximum rates of gross O₂ uptake, CO₂ uptake, and maximum O₂ evolution in wild-type cells grown continuously with 2% CO₂ (A), or on the relative induction of cmpA, sbtA, and chpY transcripts in wild-type cells transferred from aeration with 2% CO₂ in air to air bubbling for 30 min (B). Transcript abundance was determined by real-time RT-PCR (n = 4), and symbols represent the extent of induction or repression after the shift to low Ci as a percentage of the 0 mm SHAM value (set at 100%) ± se. The 0 mm values (i.e. 100%) equate to average rates of gross O₂ uptake, CO₂ uptake, and maximum O₂ evolution of 270, 190, 480 μmol mg⁻¹ h⁻¹, respectively (n ≥ 3), and approximately 55-fold, 65-fold, or 7-fold increases in cmpA, sbtA, and chpY transcript pools, respectively.