PsfR, a factor that stimulates psbAI expression in the cyanobacterium Synechococcus elongatus PCC 7942
24 Proteins 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
... 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 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 Mathee, 1998; Paget, 2015). 69 In this study, we identified a candidate for the principal sigma factor binding protein 70 among cyanobacteria and confirmed the interaction in a cyanobacterium Synechococcus 71 elongatus PCC 7942 (hereafter S. elongatus). Cyanobacteria harbor multiple alternative 72 sigma factors, including several group 2 sigma factors, to help to respond to various 73 environmental stresses (Osanai et al., 2008). A role of the newly-identified factor for the 74 sigma factors substitution and the heat-stress responsive transcriptional regulation is 75 discussed. 76 77 Materials and Methods 78 Bacterial strains, culture conditions, and recombinant DNA techniques. 79 S. elongatus and mutants were grown in BG-11 (Rippka, 1988) liquid medium or on 80 BG-11 plate containing 1.5% w/v agar at 30°C with 2% CO 2 aeration under constitutive 81 fluorescent light (30 µmol photons/m 2 /s). BG-11 plates were supplemented with 40 82 µg/mL spectinomycin or 10 µg/mL kanamycin as required. E. coli DH5α was used for 83 all cloning procedures using standard techniques. PCR reactions were performed using 84 KOD-Plus-Neo (Toyobo Co., Ltd., Osaka, Japan). Oligonucleotide primers were 85 commercially purchased from a supplier (Integrated DNA Technologies, Inc., Coralville, 86 IA, USA). Detailed procedures for the construction of plasmids and strains are 87 described in Supplemental Materials and Methods. 88 89 Phylogenetic analysis. 90 The amino acid (aa) sequences of ten SinA homologs were identified based on 91 CyanoBase (Nakamura et al., 1998) and GenBank (Benson et al., 2009) and analyzed 92 using ClustalW (Thompson et al., 1994). The unrooted phylogenic tree was constructed 93 using the neighbor-joining method (Saitou and Nei, 1987) with MEGA7 (Kumar et al., 94 2016) based on the multiple sequence alignments. The bootstrap procedure sampled 95 1,000 times with replacement with MEGA7 (Kumar et al., 2016) to estimate the 96 reliability of the tree. 97 98 Cell viability test. 99 The cell suspensions were serially diluted in 10-fold intervals with BG-11 medium, and 100 a 7 µL aliquot from each dilution was spotted onto BG-11 plates. Plates were then 101 incubated under illumination at a high temperature (40°C) for 7 days before being 102 photographed. 103 104 Temperature upshift experiment. 105 Cultures were grown to OD 750 = 0.5 in BG-11 liquid medium and the temperature 106 upshifts were performed by moving the cultures to a water bath (45°C or 48°C). Cells 107 were collected using centrifugation (7,000 × g, 2 min, 4°C), at 0, 5, 15 and 60 min, 108 respectively, after temperature upshifts. 109 110 RNA analysis. 111 The total RNA was extracted from cells using the hot phenol method and 5 µg RNA 112 was subjected to a Northern hybridization analysis (Seki et al., 2007). A DNA probe for 113 hspA was digoxigenin-labeled using PCR amplification with the primers [hspA-Fw and 114 hspA-Rv]. 115 116 157 histidine kinase gene hik2 in a head-to-head manner (Fig. S1), which may indicate the 158 functional relationship. 159 SinA aa sequences from several cyanobacterial strains were aligned and their 160 characteristics were compared (Fig. 2 A). A conserved Pfam domain (Finn et al., 2006) 161 of unknown function, DUF3155, was the main part of these proteins. SinA of S. 162 elongatus (Synpcc7942_0452) and Gloeobacter violaceus PCC 7421 (Gll0064) were 163 annotated in CyanoBase (Nakamura et al., 1998), however, the alignment analysis 164 revealed the N-terminal 36 aa and 39 aa regions of these SinA proteins, respectively, do 325 326 Hanaoka, M., and Tanaka, K. (2008) Dynamics of RpaB-promoter interaction during 327 high light stress, revealed by chromatin immunoprecipitation (ChIP) analysis 328 in Synechococcus elongatus PCC 7942. Plant J., 56, 327-335. 329 330 Helmann, J. D. (2002) The extracytoplasmic function (ECF) sigma factors. Adv. Microb. 331 Physiol., 46, 47-110. 332 333 Helmann, J. D., and Chamberlin, M. J. (1988) Structure and function of bacterial sigma 334 factors. Ann. Rev.