Here, we developed a method for measuring the in vivo redox state of the plastoquinone (PQ) pool in the cyanobacteria Synechocystis sp. PCC 6803. Cells were illuminated on a glass fiber filter, PQ was extracted with ethyl acetate and determined with HPLC. Control samples with fully oxidized and reduced photoactive PQ pool were prepared by far-red and high light treatments, respectively, or by blocking the photosynthetic electron transfer chemically before or after PQ in moderate light. The photoactive pool comprised 50% of total PQ. We find that the PQ pool of cyanobacteria behaves under light treatments qualitatively similarly as in plant chloroplasts, is less reduced during growth under high than under ambient CO 2 and remains partly reduced in darkness.
24Proteins that bind to RNA polymerase (RNAP) sigma factors play important 25 roles in various transcriptional regulations. In this study, we identified a candidate of the 26 principal sigma factor interacting protein in cyanobacteria, named SinA, based on a 27 previous comprehensive protein interaction study (Sato et al., 2007) and analyzed this in 28 the cyanobacterium Synechococcus elongatus PCC 7942. SinA is highly conserved 29 among cyanobacteria and a knock out mutant showed defective growth at a usually 30 permissive high temperature (40°C). Because this observation suggested SinA 31 involvement in heat-inducible transcriptional activation, we examined heat-inducible 32 protein gene hspA expression after temperature upshifts. The second-step induction 33 disappeared after 15 min in the sinA mutant. In vivo pull-down experiments 34 demonstrated the interaction between SinA and the principal sigma factor RpoD1. This 35 SinA-RpoD1 complex was associated with an RNAP core enzyme under growth 36 temperatures, but was dissociated after a temperature upshift. Based on these results, we 37 propose a function of SinA to facilitate the substitution of the principal sigma factor 38 with alternative sigma factors under heat-stressed conditions. 39 40 Abbreviations: aa, amino acid(s); ORF, open reading frame; RNAP, RNA polymerase 41 42 48 et al., 2008). 49Sigma factor is a subunit of RNAP that binds and donates promoter recognition 50 specificity and the ability to initiate transcription in bacteria to the catalytic core RNAP 51 complex (Gruber and Gross, 2003). Most bacteria encode multiple sigma factor species 52 in their genome and differential use and the promoter recognition specificity of these 53 sigma factors often explain differential gene expression from the genome depending on 54 internal and external conditions (Gruber and Gross, 2003; Sharma and Chatterji, 2010). 55 Sigma factors are categorized into several groups. The principle, or group 1, sigma 56 factor is a unique and essential sigma factor in each bacterium and is responsible for the 57 transcription of most housekeeping genes under physiological growth conditions. Group 58 2 sigma factors share a highly homologous structure with group 1 sigma factors but are 59 not essential for cell viability. Other sigma factors are categorized as group 3 sigma 60 factors, while group 4 sigma factors were proposed as extracytoplasmic function sigma 61 factors (Helmann, 2002). Each alternative sigma factor is activated under various 62 stressed conditions and is responsible for activating relevant stress responsive genes 63 (Osanai et al., 2008). 64 Sigma factor binding proteins exist in various bacteria and modulate sigma factor 65 activity through their interactions (Güell et al., 2011; Hughes and Mathee, 1998; Paget, 66 2015). Well-known examples are anti-sigma factors that inactivate sigma factor 67 functions; interaction between sigma and anti-sigma factors prevents sigma-core RNAP 68 association and downstream gene expression (Hughes and ...
The highly conserved Hik2-Rre1 two-component system is a multi-stress responsive signal-transducing module that controls the expression of hsp and other genes in cyanobacteria. Previously, we found in Synechococcus elongatus PCC 7942 that the heat-inducible phosphorylation of Rre1 was alleviated in a hik34 mutant, suggesting that Hik34 positively regulates signalling. In this study, we examined the growth of the hik34 deletion mutant in detail, and newly identified suppressor mutations located in rre1 or sasA gene negating the phenotype. Subsequent analyses indicated that heat-inducible Rre1 phosphorylation is dependent on Hik2 and that Hik34 modulates this Hik2-dependent response. In the following part of this study, we focused on the mechanism to control the Hik2 activity. Other recent studies reported that Hik2 activity is regulated by the redox status of plastoquinone (PQ) through the 3Fe-4S cluster attached to the GAF domain. Consistent with this, Rre1 phosphorylation occurred after addition of 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone (DBMIB) but not after addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) to the culture medium, which corresponded to PQ-reducing or -oxidising conditions, respectively, suggesting that the Hik2-to-Rre1 phosphotransfer was activated under PQ-reducing conditions. However, there was no correlation between the measured PQ redox status and Rre1 phosphorylation during the temperature upshift. Therefore, changes in the PQ redox status are not the direct reason for the heat inducible Rre1 phosphorylation, while some redox regulation is likely involved as oxidation events dependent on 2,6-dichloro-1,4-benzoquinone (DCBQ) prevented heat-inducible Rre1 phosphorylation. On the basis of these results, we propose a model for the control of Hik2-dependent Rre1 phosphorylation.
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