Identification and analysis of a principal sigma factor interacting protein SinA, essential for growth at high temperatures in a cyanobacterium <i>Synechococcus elongatus</i> PCC 7942

Abstract

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 ...

Retracted: The circadian rhythm regulator RpaA modulates photosynthetic electron transport and alters the preferable temperature range for growth in a cyanobacterium

“…The synpcc7942_0094 gene region was amplified from PCC7942 genomic DNA using the 0094-SalI-R/ 0094-KpnI-F primer set. The rpaA-FLAG fragment and the synpcc7942_0094 fragment were cloned into the SacI/ XbaI site and the SalI/KpnI site of pBΩH [19], respectively. The resulting pAFΩdn was used to transform PCC7942, with transformants identified based on spectinomycin resistance, to obtain the AF mutant strain.…”

Structure of the SigF1-dependent <i>pilA1</i> gene promoter and characterization of the light-activated response in the cyanobacterium <i>Synechococcus elongatus</i> PCC 7942

“…All cloning procedures were performed in E. coli DH5α following standard molecular biology protocols. To obtain a plasmid containing a FLAG epitope tag and Ω cassette (Prentki and Krisch, 1984), the psinAFΩup plasmid (Hasegawa et al, 2020) was digested with BamHI and SalI. This fragment was inserted into the BamHI/SalI site of pBluescriptSK+ (Agilent Technologies Japan, Ltd., Tokyo, Japan), yielding plasmid pFLAGΩSp.…”

A high-temperature sensitivity of <i>Synechococcus elongatus</i> PCC 7942 due to a tRNA-Leu mutation

“…Sequences of about 700 bases upstream and downstream of synpcc7942_1640 (nynA) were amplified by PCR with the primer pairs [1640up-F,1640up-R-toSp] and [1640dn-F-fromSp, 1640dn-R], respectively (Table S1). The spectinomycin resistance cassette (Sp R ) was amplified by PCR from pBΩH (Hasegawa et al, 2020) as the template with the primer pair [Sp-F-to1640, Sp-R-to1640] (Table S1). These three fragments were combined by in-fusion PCR, and the combined DNA fragment (nynA::Sp R ) was used to transform WT and H1 to obtain TDN and HDN, respectively.…”