Uncovering a novel component in the cyanobacterial carbon concentration mechanism

Cyanobacteria are key players in global biogeochemical cycles as they are responsible for at least 25% of oceanic carbon fixation (Sánchez-Baracaldo et al. 2022). Fixation of carbon dioxide (CO₂) takes place in the Calvin cycle catalyzed by Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase). Yet Rubisco faces many difficulties in today's atmosphere—it has low specificity for CO₂ and, due to higher concentrations of atmospheric oxygen than carbon dioxide, frequently catalyzes an undesired reaction with oxygen (Lechno-Yossef et al. 2020). Cyanobacteria face additional challenges, as aquatic environments have less accessible forms of carbon, mainly HCO₃⁻ as opposed to CO₂, and the rate of diffusion is slower (Nguyen et al. 2025). Hence, the supply of inorganic carbon (Ci) in water is lower than that on land, which further limits Rubisco.

To overcome Rubisco's limitations, cyanobacteria have evolved a carbon concentrating mechanism (CCM). The CCM relies on cytoplasmic accumulation of HCO₃⁻, which diffuses into Rubisco-containing protein compartments, called carboxysomes. Inside carboxysomes HCO₃⁻ is converted to CO₂ by carbonic anhydrases, resulting in high concentrations of CO₂ around Rubisco, which improves the efficiency of carbon fixation (Nguyen et al. 2025). Uptake of Ci is a key step for the cyanobacterial CCM, so it is unsurprising that there are at least 5 uptake systems (Rottet et al. 2021). One is the sodium-dependent bicarbonate transporter A (SbtA) that is induced under low CO₂ and has a high affinity for HCO₃⁻ (Shibata et al. 2002). For many years the regulation of SbtA remained enigmatic until the identification of SbtB that may inhibit SbtA-mediated HCO₃⁻ transport and prevent HCO₃⁻ leakage during the dark (Du et al. 2014; Haffner et al. 2023). Beyond SbtB no additional proteins are known to be involved in SbtA-dependent transport, so identification of additional regulators is of great interest.

In a recent article in Plant Physiology, Walke et al. (2026) identify a previously unannotated small open reading frame (ORF) upstream of SbtA and SbtB in the model cyanobacterium Synechocystis sp. PCC 6803, which they call SbtC. Using northern blotting, they demonstrated that SbtC is transcribed under low CO₂ conditions. To confirm whether the transcript produced a functional protein, the gDNA was fused to a FLAG-tag encoding sequence and placed under a copper response promoter in Synechocystis. When copper was supplied, a FLAG-tag protein was detected via western blotting. Together, these results suggested that SbtC is responsive to low CO₂ and produces a protein.

The authors proceeded in generating a ΔsbtC mutant in Synechocystis using homologous recombination with a gentamicin resistance cassette. Generally, ΔsbtC mutants had no growth phenotype except under diurnal light and low CO₂ conditions when growth was slightly, but not significantly, reduced. This phenotype resembled that of a sbtB mutant (Selim et al. 2021). The relatively minor growth phenotypes are unsurprising given the redundancy in cyanobacterial Ci uptake mechanisms (Rottet et al. 2021). Whether the ΔsbtC mutation, or indeed a sbtB mutation, would have a stronger effect in noncontrolled conditions has not been tested.

To test whether SbtC influences Ci uptake, the authors utilized the Δ5 mutant. The Δ5 mutant lacks all Ci uptake systems and is a robust background to use for validating putative Ci uptake mechanisms (Xu et al. 2008). The SbtABC genes were re-introduced in the Δ5 mutant, and SbtB or SbtC was knocked out independently (Fig. 1). Comparative analysis of these strains revealed that SbtC inhibits SbtA-dependent Ci transport, as strains without SbtC have greater Ci uptake than strains expressing SbtC (Fig. 1a). Both SbtB and SbtC minimized Ci leakage via SbtA during periods of darkness, as strains lacking either protein had significantly greater Ci leakage (Fig. 1b). The role of SbtB in preventing leakage was unsurprising, as it had been hypothesized that SbtB plugs SbtA to prevent Ci loss (Haffner et al. 2023). How SbtC fits into this model is unclear, but perhaps it may stabilize the SbtA-SbtB interaction.

Figure 1.

The role of SbtC was deciphered using the Δ5 mutant expressing different combinations of SbtA/B/C. The Δ5 mutant lacks all Ci uptake systems. The SbtABC genes were re-introduced in the Δ5 mutant (called Δ5 with SbtA/B/C), and SbtB or SbtC was knocked out independently, called Δ5 with SbtA/C and Δ5 with SbtA/B, respectively. a) In the light SbtA/B/C takes up Ci into the cytoplasm, when SbtC is absent there is greater Ci uptake. b) In the dark there is leakage of Ci out of the cytoplasm via SbtA/B/C; this leakage is enhanced when either SbtB or SbtC is absent. In (a) and (b) Ci flux is represented by arrow size and width.

SbtA is membrane localized (Fang et al. 2021), and computational analysis suggested that SbtC may have a transmembrane helix. Therefore, the authors proposed that SbtC might stabilize SbtA by direct association in the membrane. To investigate if SbtC forms a complex with SbtA/B, membrane protein complexes from a Synechocystis strain expressing FLAG-tagged SbtC were isolated and were analyzed via blue native PAGE and western blotting. SbtA, SbtC, and SbtB proteins co-migrated, which suggested they are in the same complex. Based on expected protein sizes, the authors propose a ternary complex consisting of a SbtA trimer, SbtB trimer, and a SbtC monomer. Additional analysis is required to confirm these proteins directly interact with each other, as protein-protein interaction experiments were not conducted. Furthermore, employing structural techniques to determine the precise conformation of these proteins could provide insights into the molecular mechanism of SbtC action, as similar strategies have been used to successfully elucidate the role of SbtB (Fang et al. 2021).

All experiments conducted here were performed with Synechocystis sp. PCC 6803. Curiously, when the authors looked beyond this species, they found that not all cyanobacteria possess a SbtC homolog, suggesting that SbtC is not essential. Analysis of the presence/absence of SbtC revealed no correlation between SbtC's presence and either phylogeny or cyanobacterial lifestyle. The lack of correlation was surprising and raises several interesting questions. First, is there a selection pressure driving SbtC retention in certain species, and if so what is it? Second, does SbtC always perform the same role, or does its function vary according to factors, such as the type of environment the cyanobacteria inhabit? And third, in species that do not have SbtC, is there an alternative protein that performs its function?

In summary, this work by Walke and colleagues identifies a novel component of the SbtA/B Ci uptake system. Their study identifies the SbtC ORF that produces a small protein and affects Ci transport, minimizes Ci leakage and may stabilize the SbtA/B complex. These findings raise several important questions. For example, how is SbtC regulated and expressed in response to Ci limitation? Does SbtC have further unelucidated roles in the cyanobacterial CCM? And more broadly, how many other unannotated ORFs are also producing important proteins for the cyanobacterial CCM or other processes? If anything, this manuscript teaches us the need to revisit genome annotation and ORFs that may have previously been overlooked.

Related articles in Plant Physiology:

• Xin et al. (2026) described the acetylation of a cyanobacterial Ci uptake protein, ChpX, and how this modification is necessary for efficient CO₂ uptake.

• Sun et al. (2024) deciphered the structural organization of RuBisCo in the carboxysome of Synechococcus elongatus PCC7942.

Funding

None declared.

Data availability

No new data were generated or analyzed in support of this research.