Gene expression control of carbon assimilation in cyanobacteria - new insights into an old story?
Final Report Abstract
In the course of this project key regulators of primary metabolism in the model cyanobacterium Synechocystis sp. PCC 6803 were investigated and functionally characterized. For instance, we have characterized the transcriptional regulator encoded by the essential sll0998 gene that was assumed to control genes of the Calvin-Benson-Bassam (CBB) cycle. Transcriptomic analyses of a partial but phenotypically stable knockout strain revealed that this regulator is a crucial element to control the expression of genes encoding proteins required for the uptake, accumulation and incorporation of inorganic carbon (Ci) into cellular metabolism. Among them was the rbcLXS operon, which encodes the large and small subunits of the essential enzyme for CO2 fixation, ribulose-1,5-bisphosphatcarboxylase/-oxygenase (RuBisCO) and its assembly chaperone RbcX. Hence, we named this protein RbcR (RuBisCO regulator) in accordance with similar proteins found in other autotrophic microorganisms. RbcR binds the DNA region upstream of the rbcLXS genes by targeting the sequence motif ATTA(G/A)-N5-(C/T)TAAT. Thereby, RbcR acts as a transcriptional activator, which explains why this regulatory protein is essential for cell viability as RuBisCO cannot be deleted in cyanobacteria. Moreover, we investigated the regulatory RNA NsiR4, which accumulates under nitrogen (N) limitation and shows partial complementarity to several mRNAs encoding other key enzymes of the CBB cycle. Even though, the initial assumption that NsiR4 controls enzyme activities of primary C metabolism in a N-dependent manner could not be confirmed in Synechocystis, another gene, ssr0692, was identified as a direct target of NsiR4. To facilitate physiological conclusions of this regulation we also characterized the ssr0692 gene function, which was not known. Upon detailed investigation the small, 51 amino acid protein encoded by ssr0692 was found to accumulate in response to increased ammonium supply. It fulfills a crucial regulatory role in cyanobacterial metabolism via the interaction with the PII signaling protein. We showed that it directly interferes with the PII-dependent activation of N-acetyl glutamate kinase (NAGK), the key enzyme in the arginine synthesis pathway. Consistently, under fluctuating N regimes ssr0692 mutant strains were impaired in balancing the synthesis of arginine and other amino acids associated with the cyanobacterial ornithine ammonia cycle. We therefore named the encoded protein PII-interacting regulator of arginine synthesis (PirA). Consistently, the interaction of NsiR4 with the pirA 5’UTR clearly diminished PirA synthesis in response to ammonium, which also interfered with arginine synthesis. As NsiR4 and PirA are inversely regulated by the global nitrogen transcriptional regulator NtcA, this regulatory axis enables fine tuning of arginine synthesis and is believed to convey additional metabolic flexibility under highly fluctuating conditions that affect C/N balance. The obtained results did not only enhance our understanding of flux control in cyanobacteria, it will also help to provide a scientific fundament for targeted metabolic engineering and hence the design of photosynthesis-driven biotechnological applications.
Publications
- (2018) The distinctive regulation of the cyanobacterial glutamine synthetase. Life 8 (4): 52
Bolay P, Muro-Pastor MI, Florencio FJ, Klähn S
(See online at https://doi.org/10.3390/life8040052) - (2019) Same same but different: Wie Cyanobakterien ein zentrales Enzym regulieren. BIOspektrum 25 (6): 610–613
Klähn S
(See online at https://doi.org/10.1007/s12268-019-0208-x) - (2020) Small but smart: On the diverse role of small proteins in the regulation of cyanobacterial metabolism. Life 10 (12), 322
Brandenburg F and Klähn S
(See online at https://doi.org/10.3390/life10120322) - (2021) The novel PII-interacting protein PirA controls flux into the cyanobacterial ornithine-ammonia cycle. mBio 12:e00229-21
Bolay P, Rozhbeh R, Muro-Pastor MI, Timm S, Hagemann M, Florencio FJ, Forchhammer K, Klähn S
(See online at https://doi.org/10.1128/mbio.00229-21)
