Functional analysis of the protein IdiC (ORF5) being expressed in elevated amounts in Synechococcus elongatus PCC 7942 in the late growth phase or under iron limitation
Final Report Abstract
Iron deficiency leads substantial modifications in the photosynthetic and respiratory electron transport chain including a decrease in photosynthetic open chain electron transport and an increase in photosynthetic cyclic electron transport around PS I and respiratory activity. The cyanobacterial NDH-1 complex is involved in both, in photosynthetic cyclic electron transport as well as in respiration. The adaption to iron limitation includes also the expression of ironregulated proteins like IdiA, which is involved in iron-dependent modification of PS ll and whose expression is regulated by the positively acfing transcripfion regulator IdiB. The gene idiC, which is part ofthe iron-responsive idiB operon of S. elongatus PCC 7942, encodes for a protein showing the highest similarities to the substrate-binding subunits ofthe bacterial NDH-1 complex in E. coli. Thus, the major goal of this work was an investigation of the possible funcfion of IdiC in adaptation of photosynthesis and respiration to iron limitation and to get an insight in the regulatory network for acclimation of the photoautotrophic cyanobacterium S. elongatus PCC 7942 to iron limitation. IdiC protein might be a component ofthe long searched substrate-binding part of the cyanobacterial NDH-1 complex and thus, has indeed a function in the adaptive process leading to an up-regulation of photosynthetic cyclic and/or respiratory electron transport under iron-limiting grov\rth conditions and possibly also other stressful growth condifions. The IdiC protein belongs to the thioredoxin-like [2Fe-2S] ferredoxin family and has the highest similarity to the substrate binding subunit NuoE of the Escherichia coli NDH-1 complex. In S. elongatus PCC 7942 the IdiC protein is loosely attached to the thylakoid membrane and its expression is increased under iron starvation and in the late growth phase, representing growth conditions, which favour photosynthetic cyclic and respiratory electron transport over photosynthetic linear electron transport from water to NADP+. Both conditions are known to require the NDH-1 complex. S. elongatus PCC 7942 mutants, which expressed the recombinant IdiC protein in elevated amounts, showed a typical iron-deficient phenotype even under iron sufficient growth conditions indicating the loss of iron caused by high rates of IdiC synthesis. In addition, spectroscopic analyses revealed an iron-binding character of heterologously expressed IdiC protein and provided evidence for the presence of a yet unknown kind of [Fe-S] centre, which fits the observation that it becomes expressed under iron starvation. Since it was not possible to construct an IdiC knock-out mutant, IdiC seems to be essential for the viability of S. elongatus PCC 7942 under the used experimental conditions. The comparison of an IdiC-merodiploid S. elongatus PCC 7942 mutant (MuD) with S. elongatus PCC 7942 wild type (WT) revealed major differences in the adapfion to iron-deficient growth conditions. Iron-straved MuD cells showed a highly reduced growth rate, chlorophyll content, and photosynthetic O2 evolving activity than WT. In addition the respiratory activity based on the endogenous substrates was substantially lower in MuD than in WT indicafing a lower activity ofthe NDH-1 complex as a result of a reduced IdiC content. As a consequence of a reduced content of the transcripfion factor IdiB in MuD, IdiA and IrpAB expression were also decreased. Due to an impaired adaptational process to ironlimitation the redox state of components of the photosynthetic electron transport chain differ to that of WT. Therefore, the IsiA protein concentration was lower in MuD than in WT, although the isiA mRNA was equally high indicating a posttranscriptional regulation of IsiA expression, which is suggested fo be regulated in parts due to the redox state ofthe plastoqinone pool. In addition, a lower expression of the ferredoxin: NADP+ oxidoreductase in mutant MuD under iron limitation compared to that of WT indicated that the cyclic electron transport is not as effecfiy facilitated in MuD as in WT. Investigations ofthe general adaptational response to iron limitation in S. elongatus PCC 7942 using DNA microarray analyses showed that six chromosome regions containing several iron-regulated genes in sequence are present in this organism. These are the irpAB region and the fut region, both encoding putative iron acquisition systems, the suf region being assumed to participate in [Fe-S] cluster assembly under oxidative stress and iron limitation, the isiAB region encoding CP43' and flavodoxin, the idiCB operon, encoding a NuoE-like protein and the transcripfion factor for idiA, and the acetate kinase regulon. Since the invesfigations included the response to iron limitation of MuD and the IdiB-free S. elongatus PCC 7942 mutant K10 it was possible to identify six genes, which seem to be additionally regulated by IdiB. Moreover, in addition to the reduced amount of IdiB in MuD, the low amount of IdiC is responsible for a number of additional changes in the abundance of mainly photosynthesis-related transcripts as compared to the WT and mutant K10. This fact may also explain why it was impossible to obtain a fully segregated IdiC-free mutant, whereas it was possible to create a fully segregated IdiB-free mutant. Furthermore, two other novel iron starvation induced regulatory genes, merR, encoding a transcriptional regulator of the Crp-family and a gene encoding a Fur-like regulator with similarifies to the PerR-family of transcripfional regulators, named furll. Investigations of a Furll knock-out mutant provided evidence that this Fur homologue protein is not involved in regulation oft he mainly iron responsive genes, but imply a role of Furil in the transcriptional regulation of the oxidative stress response in S. elongatus PCC 7942.
Publications
- (2007) Characterization of the putative iron sulfur protein IdiC (ORF5) in Synechococcus elongatus PCC 7942. Photosynth Res. 2007 Oct;94(1):91-108. Epub 2007 Aug 10
Pietsch D, Staiger D, Pistorius EK, Michel KP
- (2008) Transcript profiling reveals new insights into the acclimation of the mesophilic freshwater cyanobacterium Synechococcus elongatus PCC 7942 to iron starvation. Plant Physiol. 2008 Jun;147(2):747-63. Epub 2008 Apr 18
Nodop A, Pietsch D, Höcker R, Becker A, Pistorius EK, Forchhammer K, Michel KP
