Differential requirement of two homologous proteins encoded by sll1214 and sll1874 for the reaction of Mg protoporphyrin monomethylester oxidative cyclase under aerobic and micro-oxic growth conditions

Abstract

The two open reading frames in the Synechocystis sp. PCC 6803 genome, sll1214 and sll1874, here designated cycI and cycII, respectively, encode similar proteins, which are involved in the Mg protoporphyrin monomethylester (MgProtoME) cyclase reaction. The impairment of tetrapyrrole biosynthesis was examined by separate inactivation of both cyclase encoding genes followed by analysis of chlorophyll contents, MgProtoME levels and several enzyme activities of tetrapyrrole biosynthesis. We additionally addressed the question, whether the two isoforms can complement cyclase deficiency under normal aerobic and micro-oxic growth conditions in light. A cycII knock-out mutant grew without any adverse symptoms at normal air conditions, but showed MgProtoME accumulation at growth under low oxygen conditions. A complete deletion of cycI failed in spite of mixotrophic growth and low light at both ambient and low oxygen, but resulted in accumulation of 150 and 28 times more MgProtoME, respectively, and circa 60% of the wild-type chlorophyll content. The CycI deficiency induced a feedback-controlled limitation of the metabolic flow in the tetrapyrrole biosynthetic pathway by reduced ALA synthesis and Fe chelatase activity. Ectopic expression of the CycI protein restored the wild-type phenotype in cycI(-) mutant cells under ambient air as well as micro-oxic growth conditions. Overexpressed CycII protein could not compensate for cycI(-) mutation under micro-oxic and aerobic growth conditions, but complemented the cycII knock-out mutant as indicated by wild-type MgProtoME and chlorophyll levels. Our findings indicate the essential contribution of CycI to the cyclase reaction at ambient and low oxygen conditions, while low oxygen conditions additionally require CycII for the cyclase activity.

Conserved Chloroplast Open-reading Frame ycf54 Is Required for Activity of the Magnesium Protoporphyrin Monomethylester Oxidative Cyclase in Synechocystis PCC 6803

“…A reciprocal pulldown assay using FLAG-Ycf54 showed that Sll1214 is trapped as prey. The absence of Sll1874 in the FLAGYcf54 eluate is not so surprising bearing in mind the aerobic growth conditions used to prepare the culture of FLAG-ycf54 strain; Sll1874 is specifically expressed under micro-oxic conditions, and its level is probably minimal under atmospheric oxygen level (20,21). In contrast to the membrane-bound Sll1214/Sll1874, Ycf54 appears to be a rather hydrophilic protein (Fig.…”

“…Previous work had identified two genes in Synechocystis, sll1214 and sll1874, as acsF homologs that encode the membrane component of the MgPME cyclase (20,21). To identify their protein partners, we tagged the N termini of Sll1214 and Sll1874 in vivo with 3ϫFLAG and purified both proteins from detergent-solubilized membrane extracts.…”

“…AcsF homologs have since been identified in many photosynthetic eukaryotes including C. reinhardtii (18), Arabidopsis thaliana (19), and barley (15). Two acsF-like genes, sll1214 and sll1874, have been identified in Synechocystis PCC 6803 (hereafter Synechocystis) (20); Sll1214 is essential for growth under aerobic conditions (20) and, to a certain extent, micro-oxic conditions (21), whereas Sll1874 was found to be essential for growth in micro-oxic conditions (20,21).…”

Three classes of oxygen-dependent cyclase involved in chlorophyll and bacteriochlorophyll biosynthesis

“…2A). Deletion of the native CycIencoding gene (sll1214) was attempted in this acsF Rg+ background; a previous attempt to delete cycI in the wild type (WT) under oxic conditions proved unsuccessful (19). Full segregation of ΔcycI in acsF Rg+ was achieved (Fig.…”

“…The cyanobacterium Synechocystis sp. PCC 6803 (hereinafter Synechocystis) also contains two isoforms of AcsF, designated CycI and CycII (19). Constitutively expressed cycI encodes the sole AcsF protein responsible for cyclase activity under oxic conditions, with cycII expressed only under microoxic conditions (13); overexpression of cycII cannot rescue cyclase function under a range of O 2 tensions when CycI levels are depleted (19).…”