How Small Proteins Adjust the Metabolism of Cyanobacteria Under Stress: The Role of Small Proteins in Cyanobacterial Stress Responses

Abstract

Several recently discovered small proteins of less than 100 amino acids control important, but sometimes surprising, steps in the metabolism of cyanobacteria. There is mounting evidence that a large number of small protein genes have also been overlooked in the genome annotation of many other microorganisms. Although too short for enzymatic activity, their functional characterization has frequently revealed the involvement in processes such as signaling and sensing, interspecies communication, stress responses, metabolism, regulation of transcription and translation, and in the formation of multisubunit protein complexes. Cyanobacteria are the only prokaryotes that perform oxygenic photosynthesis. They thrive under a wide variety of conditions as long as there is light and must cope with dynamic changes in the environment. To acclimate to these fluctuations, frequently small regulatory proteins become expressed that target key enzymes and metabolic processes. The consequences of their actions are profound and can even impact the surrounding microbiome. This review highlights the diverse functions of recently discovered small proteins that control cyanobacterial metabolism. It also addresses why many of these proteins have been overlooked so far and explores the potential for implementing metabolic engineering strategies to improve the use of cyanobacteria in biotechnological applications.

Keywords: biotechnology; cyanobacteria; energy metabolism; metabolic regulation; photosynthesis; small proteins; stress acclimation.

FIGURE 1

Schematic overview of problems and challenges that have resulted in small proteins frequently being overlooked. Bioinformatic challenges, as well as limitations in biochemical analyses, have contributed to the frequent neglect of small proteins in research. Bioinformatic tools used for gene prediction and protein annotation are generally optimized for larger proteins and fail in the identification of protein‐coding potential (nested genes) within larger reading frames (ORFs), or in atypical locations such as in antisense transcripts. Some mRNAs were initially classified as non‐coding RNA (often used as a collective term for antisense RNAs, sRNAs, guide RNAs, and other types of transcripts lacking coding potential), or not detected at all due to the atypically short coding sequences and the lack of conserved domains. During heterologous expression, small proteins are often only weakly expressed, secreted into the medium, or are toxic to their host cells. Because of their small size, amino acid composition, and low abundance of detectable peptides, small proteins are often difficult to stain or resolve in protein gels and can be easily missed in mass spectrometry (MS) analyses.

FIGURE 2

Nitrogen assimilation and carbon metabolism are intimately intertwined in cyanobacteria (e.g., Synechocystis). The main nitrogen uptake systems in cyanobacteria are the ABC‐like nitrate/nitrite bispecific transporter (NRT), the ammonia permease (AMT), and urea uptake system (URT). The most preferred nitrogen source is ammonia (NH₄ ⁺). Urea is converted to NH₄ ⁺ by urease, whereas nitrate (NO₃ ⁻) is reduced to nitrite (NO₂ ⁻) by nitrate reductase (NR) followed by a second reduction to NH₄ ⁺ by the ferredoxin‐nitrite reductase (NiR). NH₄ ⁺ is incorporated into glutamate (Glu) to produce glutamine (Gln) by the enzymes glutamine synthetase (GS) and 2‐oxoglutarate‐aminotransferase (GOGAT) in the GS/GOGAT cycle. The main carbon uptake systems are the bicarbonate (HCO₃ ⁻) transport system BicA, the sodium‐dependent, high‐affinity bicarbonate transporter SbtA, and the ABC‐type bicarbonate transporter BTC, composed of proteins CmpABCD. The enzyme ribulose bisphosphate carboxylase (RuBisCO) fixes CO₂ in a specialized microcompartment, the carboxysome. This is an essential step in the Calvin–Benson–Bassham (CBB) cycle to produce 3‐phosphoglycerate (3‐PGA). 3‐PGA is converted to 2‐PGA and phosphoenolpyruvate (PEP) and used in the tricarboxylic acid (TCA) cycle. In cyanobacteria, 2‐oxoglutarate (2‐OG) serves as a bridge between the nitrogen assimilation via the GS/GOGAT cycle and carbon metabolism via the CBB and TCA cycles.

FIGURE 3

The signaling protein P_II controls the activity of multiple different enzymes and regulators involved in nitrogen and carbon metabolism through protein–protein interactions with small proteins. (A) The P_II interacting protein X (PipX) binds the global transcription factor NtcA to form an active complex. Two PipX proteins bind one 2‐oxoglutarate (2‐OG)‐activated NtcA dimer. Depending on the location of the 14 nt long NtcA binding sites within the respective promoters, transcription, and subsequent protein production is activated or inhibited under different conditions. Binding to a position centered close to position −41.5 upstream of the transcription start site (TSS) activates the gene expression. Conversely, binding to other sites or a motif overlapping the TSS inhibits gene expression. (B) The P_II interacting regulator of arginine synthesis (PirA) accumulates during ammonia upshifts. It competes with N‐acetyl‐l‐glutamate kinase (NAGK) for binding P_II, leading to the release of free NAGK and the repression of arginine synthesis. (C) The P_II‐interacting regulator of carbon metabolism (PirC) is upregulated during chlorosis and controls the carbon flux from the CBB to the TCA cycle. Depending on the carbon/nitrogen balance and 2‐OG level, PirC is activated by the release of P_II and inhibits the glycolytic phosphoglycerate mutase (PGAM) reaction, converting 3‐PGA into 2‐PGA.

FIGURE 4

Small proteins play essential roles in stabilizing and regulating photosynthetic complexes and the ATP synthase. (A) Schematic representation of small proteins as components of large supercomplexes within the photosynthetic apparatus, highlighting their structural roles in maintaining the assembly, stability, and functional organization of these complexes. Attached to the thylakoid membrane, phycobilisomes function as light‐harvesting antennae linked to photosystem II (PSII). In PSII, water splitting generates electrons in the cyclic electron transfer (CET) and protons in the thylakoid lumen. The electron transport chain begins with the transfer of electrons to plastoquinone (PQ), reducing it to plastoquinol (PQH2). The cytochrome b₆f (cytb₆f) complex uses the PQ cycle to couple the electron transfer from PSII via PQH₂ to plastocyanin (Pc), and to photosystem I (PSI). From PSI, electrons are transferred to ferredoxin (Fd), and subsequently to ferredoxin‐NADP⁺ reductase (FNR), reducing NADP⁺ to NADPH. The proton gradient generated in this process drives the ATP production through ATP synthase. (B) The light‐induced small protein SliP4 forms a dimer and functions as a linker and stabilizing protein within the NDH1‐L complex, PSI and PSII. It connects and stabilizes PSI via the NDH1‐L subunit as well as PSII and PSI, thereby ensuring the structural integrity of these complexes under high‐light conditions. (C) In the absence of light, the cyanobacterial ATP synthase inhibitory factor, AtpΘ, accumulates and likely binds to the subunits a and the c ring of the ATP synthase. This interaction effectively inhibits the rotary mechanism that is responsible for ATP production. (D) The small protein NblD is specifically expressed during nitrogen starvation from an NtcA‐responsive promoter. NblD interacts with chromophorylated CpcB subunits and plays a crucial role in the disassembly of phycobilisomes.

FIGURE 5

Small proteins involved in the regulation of carbon metabolism. (A) The small protein AcnSP acts as a modulator of aconitase within the tricarboxylic acid (TCA) cycle. By binding to aconitase, it regulates the affinity of the enzyme for citrate and thus influences its catalytic activity and the overall metabolic flow. (B) The redox‐sensitive small protein CP12 plays a regulatory role in the Calvin cycle, which is responsible for fixing CO₂. Under oxidative conditions, CP12 forms a complex with glyceraldehyde‐3‐phosphate dehydrogenase (GapDH) and phosphoribulokinase (PRK), which results in a reduction in the activities of these key enzymes and modulates the efficiency of the Calvin cycle.

FIGURE 6

Overview of the roles of small proteins in cyanobacteria with their potential impact on the microbiome. Small proteins (highlighted in blue letters) in cyanobacteria play diverse roles ranging from regulating internal metabolic processes to influencing the surrounding microbiome. Secreted metabolites of metabolic pathways controlled by small proteins can serve as products for other organisms. Thus, small proteins play a pivotal role in the formation of both internal and external metabolic networks, thereby underscoring their importance for the ecosystem and the interactions between organisms within the microbiome.