Munehiko Asayama

Purification, characterization, and gene expression of all sigma factors of RNA polymerase in a cyanobacterium

The expression of RNA polymerase (RNAP) sigma factor genes and proteins was characterized as a first step toward understanding their functions in a unicellular cyanobacterium Synechocystis sp. PCC 6803, which can perform photosynthesis. All nine sigma factors (group 1, SigA; group 2, SigB to SigE; and group 3, SigF to SigI) and each RNAP core subunit (RpoA, RpoB, RpoC1 and RpoC2) were overproduced and purified from Escherichia coli cells, then polyclonal antibodies were prepared. Western blot and primer extension analyses revealed that the intracellular levels of group 1 and 2 sigma factors ranged from 0.9 fmol to 9.3 fmol per microgram of the total protein under conditions of steady-state growth, and that growth phase-dependent or constitutive transcripts were observed. Interestingly, no group 3 sigma factor proteins were detected under normal physiological conditions whereas their transcripts were robust, implying a possible regulation of translational attenuation and/or protein instability. Phylogenetic analysis also revealed that group 3 sigma factor homologues of cyanobacteria are conserved with evolutionary or functionary divergence among them. In vitro and in vivo results indicated significant evidence of high-light responsive SigD expression and its promoter recognition of the photosynthesis gene, psbA. On the other hand, autoregulated sigB transcription, a dramatically increased SigB expression upon the exposure of cells to heat-shock, and specific promoter recognition by SigB with redundancy of other sigma factors on the heat-shock hspA promoter were observed. These findings clearly indicated that SigB is a heat-shock responsive sigma factor. The unique promoter architecture and expression of the relevant sigma factor gene are also discussed herein.

Antagonistic dark/light-induced SigB/SigD, group 2 sigma factors, expression through redox potential and their roles in cyanobacteria

The expression of group 2 sigma factors is characterized in a cyanobacterium Synechocystis sp. PCC 6803 grown in culture, changing light conditions (white, red and blue light, and darkness), or the presence of drugs (rifampicin, chloramphenicol, DCMU, and DBMIB), and the roles of these sigma factors are elucidated. The expression of dark/light‐induced SigB/SigD was accelerated under opposite redox (oxidation/reduction) states in an electron transport chain of photosynthesis. Expression of the dark‐induced lrtA and light‐induced psbA2/3 transcript was significantly reduced in the sigB and sigD knockout strains, respectively. Abundant amounts of sigB transcript and protein were observed in the sigC knockout strain, implying that SigC represses SigB expression under light. These findings clearly showed that SigB/SigD with another group 2 sigma, SigC, contribute to transcription for a subset of dark/light‐responsive genes in the cyanobacterium. A possible model for SigB/SigD is presented and the potential ability for promoter recognition is also discussed.

Sigma Factors for Cyanobacterial Transcription

Cyanobacteria are photosynthesizing microorganisms that can be used as a model for analyzing gene expression. The expression of genes involves transcription and translation. Transcription is performed by the RNA polymerase (RNAP) holoenzyme, comprising a core enzyme and a sigma (σ) factor which confers promoter selectivity. The unique structure, expression, and function of cyanobacterial σ factors (and RNAP core subunits) are summarized here based on studies, reported previously. The types of promoter recognized by the σ factors are also discussed with regard to transcriptional regulation.

ChlH, the H subunit of the Mg-chelatase, is an anti-sigma factor for SigE in Synechocystis sp. PCC 6803

Although regulation of sigma factors has been intensively investigated, anti-sigma factors have not been identified in oxygenic photosynthetic organisms. A previous study suggested that the sigma factor, SigE, of the cyanobacterium Synechocystis sp. PCC 6803, a positive regulator of sugar catabolism, is posttranslationally activated by light-to-dark transition. In the present study, we found that the H subunit of Mg-chelatase ChlH interacts with sigma factor SigE by yeast two-hybrid screening, and immunoprecipitation analysis revealed that ChlH associates with SigE in a light-dependent manner in vivo. We also found that Mg2+ promotes the interaction of SigE and ChlH and determines their localization in vitro. In vitro transcription analysis demonstrated that ChlH inhibits the transcription activity of SigE. Based on these results, we propose a model in which ChlH functions as an anti-sigma factor, transducing light signals to SigE in a process mediated by Mg2+.

Growth Phase-dependent Activation of Nitrogen-related Genes by a Control Network of Group 1 and Group 2 σ Factors in a Cyanobacterium

It has been reported that an RNA polymerase σ factor, SigC, mainly contributes to specific transcription from the promoter PglnB-54,-53 under nitrogen-deprived conditions during the stationary phase of cell growth in the cyanobacterium Synechocystis sp. strain PCC 6803 (Asayama, M., Imamura, S., Yoshihara, S., Miyazaki, A., Yoshida, N., Sazuka, T., Kaneko, T., Ohara, O., Tabata, S., Osanai, T., Tanaka, K., Takahashi, H., and Shirai, M. (2004) Biosci. Biotechnol. Biochem. 68, 477-487). In this study, we further examined the functions of group 2 σ factors of RNA polymerase in NtcA-dependent nitrogen-related gene expression in PCC 6803. Results indicated that SigB and SigC contribute to the transcription from PglnB-54,-53 with a σ factor replaced in a growth phase-dependent manner. We also confirmed the contribution of SigB and SigC to the transcription of other NtcA-dependent genes, glnA, sigE, and amt1, as in the case of glnB. On the other hand, the transcription of glnN was dependent on SigB and SigE. In the SigB and SigC-based regulation, the level of SigB increased, but that of SigC was constant under conditions of nitrogen deprivation. Furthermore, it was found that SigC negatively and positively regulates the level of SigB in the log and stationary phase, respectively. SigC also had a positive effect on the level of sigB transcript during the stationary phase. In contrast, SigB acts positively on SigC levels in both growth phases. These results and previous findings indicated that multiple group 2 σ factors take part in the control of NtcA-dependent nitrogen-related gene expression in cooperation with a group 1 σ factor, SigA.

SigC, the Group 2 Sigma Factor of RNA Polymerase, Contributes to the Late-stage Gene Expression and Nitrogen Promoter Recognition in the Cyanobacterium Synechocystis sp. Strain PCC 6803

We examined the role of SigC (Sll0184), a sigma factor of RNA polymerase (RNAP), in a unicellular cyanobacterium, Synechocystis sp. strain PCC 6803. On the inactivation of sigC, which is an Escherichia coli rpoD homolog, cells were viable but had a low survival rate in the stationary phase of growth under normal physiological conditions, indicating that SigC is a group 2 type sigma factor. In analyses of transcript and protein levels using the sigC knockout strain, it was found that expression of glnB, a nitrogen key regulatory gene, is controlled by SigC in the stationary phase. Primer extension revealed that the glnB nitrogen promoter (P2) was specifically recognized by SigC in the stationary phase under conditions of nitrogen starvation. In vitro studies with purified enzymes indicated effective transcription, on supercoiled DNA templates, from P2 by SigC-RNAP with NtcA which is an activator for nitrogen gene transcription. DNase I footprinting also indicated binding and related sites of NtcA and/or RNAP with SigC on the nitrogen promoter. The unique promoter architecture and the mechanism of transcription by RNAP with SigC are also discussed.

Heat shock protein 90 inhibitor 17‐allylamino‐17‐demethoxygeldanamycin potentiates the radiation response of tumor cells grown as monolayer cultures and spheroids by inducing apoptosis

Activation of the PI3K‐Akt pathway is known to induce tumor radioresistance. In the current study, we examined the ability of 17AAG, which decreases the levels of Hsp90 client proteins including components of the PI3K‐Akt pathway, to sensitize radioresistant human squamous cell carcinoma cells to X‐irradiation. Human squamous cell carcinoma cell lines (SQ20B, SCC61 and SCC13) were incubated for 16 h at 37°C in medium containing 17AAG. Radiation sensitivity was determined by clonogenic assays, and protein levels were examined by western blotting. Apoptosis was determined in monolayer cells by AO/EB double staining and in spheroids using the TdT‐mediated dUTP nick end labeling assay. 17AAG (0.2 µM) enhanced the radiosensitivity more effectively in radioresistant SQ20B and SCC13 cells than in radiosensitive SCC61 cells. However, in all three cell lines, 17AAG increased radiation‐induced apoptosis by reducing the expression of EGFR and ErbB‐2 and inhibiting the phosphorylation of Akt. Furthermore, 17AAG (1 µM) sensitized SQ20B spheroids to radiation, and inhibition of Akt activation by 17AAG increased radiation‐induced apoptosis in spheroids. The findings suggest that 17AAG effectively sensitizes radioresistant cells to radiation by inhibiting the PI3K‐Akt pathway. Targeting the PI3K‐Akt pathway with 17AAG could be a useful strategy for radiosensitization of carcinomas. (Cancer Sci 2005; 96: 911–917)

Characteristics of DNA and Multiple rpoD Homologs of Microcystis(Synechocystis) Strains

The base compositions of DNAs from nine Microcystis strains, as determined by high-performance liquid chromatography, were 41 to 42 mol% G+C. Chromosomal DNAs derived from these strains were found to be extremely resistant to many restriction endonucleases, and a restriction analysis revealed the presence of a dam-like methylase or both dam- and dcm-like methylases in all of the strains examined. Genomic Southern hybridization in which a synthetic oligonucleotide probe (rpoD probe) was used showed that members of the genus Microcystis might have multiple rpoD homologs, and the hybridization signal patterns observed with the DNAs of Microcystis aeruginosa strains were different from each other.

Specific recognition of the cyanobacterial psbA promoter by RNA polymerases containing principal sigma factors

The psbA2 gene of a unicellular cyanobacterium, Microcystis aeruginosa K-81, encodes a D1 protein homolog in the reaction center of photosynthetic Photosystem II. To clarify the promoter recognition by a sigma factor of RNA polymerase, in vivo and in vitro analyses were performed for the photosynthetic gene. Although the specific transcript from the psbA2 promoter, whose sequence is of Escherichia coli consensus type, was observed in both cyanobacterium K-81 and E. coli cells, the expression was light-dependent in K-81 whereas it was constitutive in E. coli under the conditions of light and darkness (L/D). The specific psbA2-dependent transcripts were also detected in vitro by RNA polymerases containing the principal sigma factors, E. coli sigma70 and K-81 sigmaA1 (constitutively exists in K-81 grown under L/D cycles). Furthermore, a series of promoter fragments were constructed to confirm minimal cis elements for the in vitro psbA2 transcription. A -80 to +6 or -38 to +46 region, the sequences of which consisted of a core promoter (-38 to +6), was identified as the potential minimal cis element using the RNA polymerase fraction (*EsigmaA1) containing sigmaA1 partially purified from K-81. These results suggest that the psbA2 transcription with the minimal sequence was induced by the RNA polymerase (EsigmaA1) containing the principal sigma factor, sigmaA1, under both light and dark conditions in K-81.

Dark-induced mRNA instability involves RNase E/G-type endoribonuclease cleavage at the AU-box and SD sequences in cyanobacteria

Light-responsive gene expression is crucial to photosynthesizing organisms. Here, we studied functions of cis-elements (AU-box and SD sequences) and a trans-acting factor (ribonuclease, RNase) in light-responsive expression in cyanobacteria. The results indicated that AU-rich nucleotides with an AU-box, UAAAUAAA, just upstream from an SD confer instability on the mRNA under darkness. An RNase E/G homologue, Slr1129, of the cyanobacterium Synechocystis sp. strain PCC 6803 was purified and confirmed capable of endoribonucleolytic cleavage at the AU- (or AG)-rich sequences in vitro. The cleavage depends on the primary target sequence and secondary structure of the mRNA. Complementation tests using Escherichia coli rne/rng mutants showed that Slr1129 fulfilled the functions of both the RNase E and RNase G. An analysis of systematic mutations in the AU-box and SD sequences showed that the cis-elements also affect significantly mRNA stability in light-responsive genes. These results strongly suggested that dark-induced mRNA instability involves RNase E/G-type cleavage at the AU-box and SD sequences in cyanobacteria. The mechanical impact and a possible common mechanism with RNases for light-responsive gene expression are discussed.