Cyanobacterial RNA polymerase: Structural features and acclimation to environmental change

Siirretty Doriast

The ω subunit of RNA polymerase is essential for thermal acclimation of cyanobacterium Synechocystis sp. PCC 6803

The rpoZ gene encodes the small omega subunit of RNA polymerase. A Delta rpoZ strain of the cyanobacterium Synechocystis sp. PCC 6803 grew well in standard conditions (constant illumination at 40 mu mol photons m(-2) s(-1); 32 degrees C; ambient CO2) but was heat sensitive and died at 40 degrees C. In the control strain, 71 genes were at least two-fold up-regulated and 91 genes down-regulated after a 24-h treatment at 40 degrees C, while in Delta rpoZ 394 genes responded to heat. Only 62 of these heat-responsive genes were similarly regulated in both strains, and 80% of heat-responsive genes were unique for Delta rpoZ. The RNA polymerase core and the primary sigma factor SigA were down-regulated in the control strain at 40 degrees C but not in Delta rpoZ. In accordance with reduced RNA polymerase content, the total RNA content of mild-heat-stress-treated cells was lower in the control strain than in Delta rpoZ. Light-saturated photosynthetic activity decreased more in Delta rpoZ than in the control strain upon mild heat stress. The amounts of photosystem II and rubisco decreased at 40 degrees C in both strains while PSI and the phycobilisome antenna protein allophycocyanin remained at the same level as in standard conditions. The phycobilisome rod proteins, phycocyanins, diminished during the heat treatment in Delta rpoZ but not in the control strain, and the nblA1 and nblA2 genes (encode NblA proteins required for phycobilisome degradation) were up-regulated only in Delta rpoZ. Our results show that the omega subunit of RNAP is essential in heat stress because it is required for heat acclimation of diverse cellular processes.</p

Characterization of Single and Double Inactivation Strains Reveals New Physiological Roles for Group 2 σ Factors in the Cyanobacterium Synechocystis sp. PCC 68031[W]

Cyanobacteria are eubacteria that perform oxygenic photosynthesis like plants. The initiation of transcription, mediated by the RNA polymerase holoenzyme, is the main determinant of gene regulation in eubacteria. The σ factor of the RNA polymerase holoenzyme is responsible for the recognition of a promoter sequence. In the cyanobacterium Synechocystis sp. PCC 6803, the primary σ factor, SigA, is essential for cell viability. The SigB, SigC, SigD, and SigE factors show significant sequence similarity with the SigA factor but are nonessential. In this study, we have used homology modeling to construct a three-dimensional model of Synechocystis RNA polymerase holoenzyme and all group 1 and 2 σ factors. According to the models, the overall three-dimensional structures of group 1 and 2 σ factors are similar, the SigB and SigD factors being the most similar ones. In addition, we have constructed a complete set of group 2 σ factor double inactivation strains, ΔsigBC, ΔsigBD, ΔsigBE, ΔsigCD, ΔsigCE, and ΔsigDE. All double mutants grow well under standard conditions, but differences are observed in stress conditions. The transition from lag phase to exponential growth is slow in the ΔsigBD strain, and all strains lacking the SigD factor were found to be sensitive to bright light. Furthermore, all group 2 σ factors were found to be involved in acclimation to salt- or sorbitol-induced osmotic stresses

Genes responding to mild heat stress in the control (CS) or ΔrpoZ strains.

<p>(A) The Venn diagram shows genes down-regulated or up-regulated in ΔrpoZ or CS upon 24-h treatment at 40°C. The gene was considered as differently regulated if log₂ of the fold change was ≤−1 or ≥1 and the P value was <0.05. The numbers inside the sectors indicate the numbers of overlapping and unique genes in different pairwise comparisons. Genes with known function that are down-regulated or up-regulated in both strains upon heat treatment are indicated, and also genes showing opposite response to heat treatment in the studied strains are included if their function is known. (B) Distribution of mild heat stress responsive genes to functional categories according to Cyanobase.</p

The ω Subunit of RNA Polymerase Is Essential for Thermal Acclimation of the Cyanobacterium <i>Synechocystis</i> Sp. PCC 6803

<div><p>The <i>rpoZ</i> gene encodes the small ω subunit of RNA polymerase. A ΔrpoZ strain of the cyanobacterium <i>Synechocystis</i> sp. PCC 6803 grew well in standard conditions (constant illumination at 40 µmol photons m⁻² s⁻¹; 32°C; ambient CO₂) but was heat sensitive and died at 40°C. In the control strain, 71 genes were at least two-fold up-regulated and 91 genes down-regulated after a 24-h treatment at 40°C, while in ΔrpoZ 394 genes responded to heat. Only 62 of these heat-responsive genes were similarly regulated in both strains, and 80% of heat-responsive genes were unique for ΔrpoZ. The RNA polymerase core and the primary σ factor SigA were down-regulated in the control strain at 40°C but not in ΔrpoZ. In accordance with reduced RNA polymerase content, the total RNA content of mild-heat-stress-treated cells was lower in the control strain than in ΔrpoZ. Light-saturated photosynthetic activity decreased more in ΔrpoZ than in the control strain upon mild heat stress. The amounts of photosystem II and rubisco decreased at 40°C in both strains while PSI and the phycobilisome antenna protein allophycocyanin remained at the same level as in standard conditions. The phycobilisome rod proteins, phycocyanins, diminished during the heat treatment in ΔrpoZ but not in the control strain, and the <i>nblA1</i> and <i>nblA2</i> genes (encode NblA proteins required for phycobilisome degradation) were up-regulated only in ΔrpoZ. Our results show that the ω subunit of RNAP is essential in heat stress because it is required for heat acclimation of diverse cellular processes.</p></div

Comparison of heat stress responsive genes in CS and ΔrpoZ.

<p>The left panel shows genes whose expression was at least two-fold up-regulated or down-regulated either in the control or ΔrpoZ strains or both upon heat treatment when the gene expression was compared to the expression of the same strain under standard growth conditions, and in addition gene expression of ΔrpoZ and CS were compared in standard growth conditions. The heat maps show log₂ fold change values (P<0.05) on the scale from −2 (blue) to 2 (red); values bigger than 2 are also shown in red and values smaller than −2 are blue. If the P value was ≥0.05, the fold change was given the value 0. Genes were arranged to categories according to Cyanobase, letters on the left indicating the same categories as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112599#pone-0112599-g002" target="_blank">Fig. 2B</a>. On the right, magnification of differently regulated genes in photosynthesis (top), regulatory functions (middle), and transport and binding proteins (bottom) is shown.</p

Contents of RNA polymerase and RNA in mild heat stress in the control and ΔrpoZ strains.

<p>Total proteins were isolated after 0, 2, 4 and 24 h treatments at 40°C, samples containing 50 µg of protein were separated with SDS-PAGE, and the amounts of the α (A), β (B), SigA (C) and ω (D) subunits of RNAP were determined by western blotting. (E) Total RNA content of cells after 24-h heat treatment. Total RNA content in 1-mL sample (OD₇₃₀ = 1) of CS (black bars) and ΔrpoZ (white bars) cell cultures incubated for 24 h at 40°C. Each result represents the mean of three biological replicates and the error bars denote SEM. A 5-µl sample of isolated RNA was separated in 1.2% agarose gel and stained with ethidium bromide to visualize rRNA.</p