# Regulation of mixotrophy in Synechocystis by a rhomboid protease

## TL;DR

This paper explores how a rhomboid protease in Synechocystis affects mixotrophy, a growth strategy combining photosynthesis and organic carbon use.

## Contribution

The study identifies a novel role for the Slr1461 rhomboid protease in regulating carbon uptake and gene expression in cyanobacteria.

## Key findings

- The Slr1461 protease mutant in Synechocystis shows impaired mixotrophic growth and reduced photosynthetic activity.
- Slr1461 influences gene expression related to CO2 import and the carbon-concentrating mechanism.
- The protease interacts with FtsH2 and affects the transcription factor NdhR under high CO2 conditions.

## Abstract

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 an incentive for 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 non-photochemical 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 that of WT under optimal light intensity. The most dramatic effect of the 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.