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

Gunnelius L, Kurkela J, Hakkila K, Koskinen S, Parikainen M, Tyystjärvi T - PLoS ONE (2014)

Bottom Line: The rpoZ gene encodes the small ω subunit of RNA polymerase.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Turku, Turku, Finland.

ABSTRACT
The rpoZ gene encodes the small ω subunit of RNA polymerase. A ΔrpoZ strain of the cyanobacterium Synechocystis 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 nblA1 and nblA2 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.

No MeSH data available.


Related in: MedlinePlus

Contents of RNA polymerase and RNA in mild heat stress in the control and ΔrpoZ strains.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.
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pone-0112599-g004: Contents of RNA polymerase and RNA in mild heat stress in the control and ΔrpoZ strains.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.

Mentions: Next we analyzed the RNAP content of cells in mild heat stress. The cells were grown under standard conditions and then transferred to 40°C for 2, 6 or 24 h. Western blots showed a clear decrease of RNAP during the high temperature treatment in CS; after one day treatment, cells had lost 45% of the RNAP core subunits α and β (Fig. 4AB). On the contrary, the ΔrpoZ strain lost less than 10% of RNAP core subunits α and β (Fig. 4AB). In addition, the amount of the primary σ factor, SigA, decreased in heat stress; after 24-h heat treatment 45% and 17% of SigA was lost in CS and ΔrpoZ, respectively (Fig. 4C). The ω subunit decreased similarly in CS as the other RNAP core subunits (Fig. 4D). In accordance with decrease of RNAP in CS, the total RNA content of CS cells decreased from 1.2 µg/ml in cultures with OD730 = 1 [10] to 0.8 µg/ml after a 24-h treatment at 40°C (Fig. 4E). In the ΔrpoZ strain, the RNA content was similar as in CS in standard conditions [10]. The RNA content of ΔrpoZ decreased only 17% during the 24-h heat treatment (Fig. 4E) suggesting that the higher RNAP content of ΔrpoZ keeps transcription in ΔrpoZ more active than in CS in mild heat stress. However, the stability of transcripts is known to vary according to environmental cues [38] and we cannot rule out the possibility that the RNA contents of CS and ΔrpoZ are affected by RNA stability at high temperatures.


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

Gunnelius L, Kurkela J, Hakkila K, Koskinen S, Parikainen M, Tyystjärvi T - PLoS ONE (2014)

Contents of RNA polymerase and RNA in mild heat stress in the control and ΔrpoZ strains.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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4227741&req=5

pone-0112599-g004: Contents of RNA polymerase and RNA in mild heat stress in the control and ΔrpoZ strains.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.
Mentions: Next we analyzed the RNAP content of cells in mild heat stress. The cells were grown under standard conditions and then transferred to 40°C for 2, 6 or 24 h. Western blots showed a clear decrease of RNAP during the high temperature treatment in CS; after one day treatment, cells had lost 45% of the RNAP core subunits α and β (Fig. 4AB). On the contrary, the ΔrpoZ strain lost less than 10% of RNAP core subunits α and β (Fig. 4AB). In addition, the amount of the primary σ factor, SigA, decreased in heat stress; after 24-h heat treatment 45% and 17% of SigA was lost in CS and ΔrpoZ, respectively (Fig. 4C). The ω subunit decreased similarly in CS as the other RNAP core subunits (Fig. 4D). In accordance with decrease of RNAP in CS, the total RNA content of CS cells decreased from 1.2 µg/ml in cultures with OD730 = 1 [10] to 0.8 µg/ml after a 24-h treatment at 40°C (Fig. 4E). In the ΔrpoZ strain, the RNA content was similar as in CS in standard conditions [10]. The RNA content of ΔrpoZ decreased only 17% during the 24-h heat treatment (Fig. 4E) suggesting that the higher RNAP content of ΔrpoZ keeps transcription in ΔrpoZ more active than in CS in mild heat stress. However, the stability of transcripts is known to vary according to environmental cues [38] and we cannot rule out the possibility that the RNA contents of CS and ΔrpoZ are affected by RNA stability at high temperatures.

Bottom Line: The rpoZ gene encodes the small ω subunit of RNA polymerase.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Turku, Turku, Finland.

ABSTRACT
The rpoZ gene encodes the small ω subunit of RNA polymerase. A ΔrpoZ strain of the cyanobacterium Synechocystis 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 nblA1 and nblA2 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.

No MeSH data available.


Related in: MedlinePlus