Regulation of mixotrophy in Synechocystis by a rhomboid protease

The intramembrane ‘rhomboid’ protease family is almost ubiquitous across evolution, with its well-conserved transmembrane domains typified in crystal structures of bacterial representatives, such as the Escherichia coli GlpG. In contrast with accumulating data on rhomboid function in higher organisms, where roles in human disease are in incentive to study, findings remain sparse about the functions and substrates of the prokaryotic enzymes, even though these provided the earliest protein structures. In particular, nothing at all is known about the rhomboid proteases of photosynthetic prokaryotes despite the importance of cyanobacteria as relatives of the progenitor of chloroplasts. Findings relating to the cyanobacterial enzymes would complement data on plant plastid rhomboids from work in Arabidopsis thaliana. Synechocystis sp. PCC 6803 was used, therefore, to investigate conserved photosynthetic functions across evolution for this protein family. Reverse-genetics studies using Slr1461, the single rhomboid protease of Synechocystis 6803, did not reveal a nonphotochemical quenching phenotype as observed for the Arabidopsis RBL10 null mutant which lacked a chloroplast outer membrane rhomboid. The Slr1461 mutant exhibited a marginal change in pigment composition and its growth rate was only slightly different from WT under optimal light intensity. The most dramatic effect of inactivation of Slr1461 was the mutant’s distinct inability to reduce photosynthetic activity under mixotrophic conditions. Concurrent phototrophy and heterotrophy in mixotrophic growth aids survival and competitiveness in phytoplankton, allowing conservation of energy by reducing the need for uptake and fixing of CO2 when an organic carbon source is available. It was notable therefore that, in the absence of the Slr1461 rhomboid, the steady-state mRNA levels were reduced for a subset of genes encoding facilitators of high-affinity CO2 import and of transcriptional regulators of the carbon-concentrating mechanism (CCM). Slr1461 activity was also linked with that of another membrane protease, the AAA protease FtsH2, which was likewise observed to act within regulatory networks for the cyanobacterial carbon uptake mechanism. Aberrant transcript levels were most evident specifically under high CO2 conditions, when the impact of Slr1461 enzymatic activity appeared to be upstream of NdhR, a central, controlling transcription factor of the CCM. Molecular biology and reverse-genetics studies were made on Slr1461, the single rhomboid protease of Synechocystis sp PCC 6803, focussing on photosynthesis and CO2 accumulation. Inactivation of slr1461 did not affect nonphotochemical quenching as seen with chloroplast RBL10, but Slr1461 was required for reduction of photosynthetic activity in mixotrophic conditions. This mechanism allows cyanobacteria to conserve energy by reducing uptake of CO2 when an organic carbon source is available: indeed, the presence of Slr1461 was linked with inhibition of genes facilitating high-affinity CO2 import under high CO2 and mixotrophic conditions. The carbon-concentrating mechanism is regulated by a network in which Slr1461 appears to be located upstream of the central, controlling transcription factor. We demonstrate that rhomboid Slr1461, in concert with another protease, FtsH2, regulates transcription of genes encoding the cyanobacterial high-affinity CO2 uptake system.