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A mixed incoherent feed-forward loop contributes to the regulation of bacterial photosynthesis genes.

Mank NN, Berghoff BA, Klug G - RNA Biol (2013)

Bottom Line: Living cells use a variety of regulatory network motifs for accurate gene expression in response to changes in their environment or during differentiation processes.In Rhodobacter sphaeroides, a complex regulatory network controls expression of photosynthesis genes to guarantee optimal energy supply on one hand and to avoid photooxidative stress on the other hand.This point-of-view provides a comparison to other described feed-forward loops and discusses the physiological relevance of PcrZ in more detail.

View Article: PubMed Central - PubMed

Affiliation: Institut für Mikrobiologie und Molekularbiologie; Universität Giessen; Giessen, Germany.

ABSTRACT
Living cells use a variety of regulatory network motifs for accurate gene expression in response to changes in their environment or during differentiation processes. In Rhodobacter sphaeroides, a complex regulatory network controls expression of photosynthesis genes to guarantee optimal energy supply on one hand and to avoid photooxidative stress on the other hand. Recently, we identified a mixed incoherent feed-forward loop comprising the transcription factor PrrA, the sRNA PcrZ and photosynthesis target genes as part of this regulatory network. This point-of-view provides a comparison to other described feed-forward loops and discusses the physiological relevance of PcrZ in more detail.

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Figure 3. Representative northern blots for pucBA and PcrZ expression kinetics after drop of oxygen tension at time point 0. R. sphaeroides 2.4.1 wild-type (A) and R. sphaeroides PcrZ overexpression strain (pRKPcrZ) (C) pre-cultures were grown overnight under high oxygen conditions (8 mg/L soluble O2) to an OD660nm of 0.6–0.8 (t0). Samples were collected at indicated time points after a shift to low oxygen conditions (0.5 mg/L soluble O2). Fifteen µg of total RNA were separated on 1% agarose gels containing 2.2 M formaldehyde. For pucBA detection, a specific DNA fragment was radioactively labeled with (α-32P)-dCTP using the NEBlot kit (New England Biolabs). A 14S rRNA-specific oligonucleotide was end-labeled with (γ-32P)-ATP and served as loading control. In R. sphaeroides, 23S rRNA is processed to 16S, 14S and 5.8S rRNA fragments by RNase III.46,47 For PcrZ and 5S rRNA, 7.5 µg of total RNA was separated on 10% polyacrylamide gels containing 7 M urea. PcrZ and 5S rRNA-specific oligonucleotides were radioactively end-labeled with (γ-32P)-ATP and were used for detection. 5S rRNA served as loading control. (B) Quantification of northern blot signals for wild-type (A). (D) Quantification of northern blot signals for pRKPcrZ (C). The mean and the standard error are based on three independent biological experiments. The Y axis to the left shows the fold change of pucBA (black squares), the Y axis to the right displays the fold change of PcrZ (black circles).
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Figure 3: Figure 3. Representative northern blots for pucBA and PcrZ expression kinetics after drop of oxygen tension at time point 0. R. sphaeroides 2.4.1 wild-type (A) and R. sphaeroides PcrZ overexpression strain (pRKPcrZ) (C) pre-cultures were grown overnight under high oxygen conditions (8 mg/L soluble O2) to an OD660nm of 0.6–0.8 (t0). Samples were collected at indicated time points after a shift to low oxygen conditions (0.5 mg/L soluble O2). Fifteen µg of total RNA were separated on 1% agarose gels containing 2.2 M formaldehyde. For pucBA detection, a specific DNA fragment was radioactively labeled with (α-32P)-dCTP using the NEBlot kit (New England Biolabs). A 14S rRNA-specific oligonucleotide was end-labeled with (γ-32P)-ATP and served as loading control. In R. sphaeroides, 23S rRNA is processed to 16S, 14S and 5.8S rRNA fragments by RNase III.46,47 For PcrZ and 5S rRNA, 7.5 µg of total RNA was separated on 10% polyacrylamide gels containing 7 M urea. PcrZ and 5S rRNA-specific oligonucleotides were radioactively end-labeled with (γ-32P)-ATP and were used for detection. 5S rRNA served as loading control. (B) Quantification of northern blot signals for wild-type (A). (D) Quantification of northern blot signals for pRKPcrZ (C). The mean and the standard error are based on three independent biological experiments. The Y axis to the left shows the fold change of pucBA (black squares), the Y axis to the right displays the fold change of PcrZ (black circles).

Mentions: Oxygen tension and light are the most important stimuli controlling formation of photosynthetic complexes in R. sphaeroides. A drop of oxygen tension is an activating stimulus and corresponding expression kinetics of photosynthetic complexes were first monitored for R. capsulatus, which shares many factors involved in regulation of photosynthesis genes with R. sphaeroides.23 In R. capsulatus, mRNA levels of photosynthesis genes intensely increased after drop of oxygen tension, reached a maximum after 30–60 min and decreased again. A strong increase of photosynthesis gene expression (e.g., pucBA) is also observable in R. sphaeroides, but maximal expression is reached only 120 min following the drop of oxygen tension. After maximal expression levels are reached, we observed only a slow decrease of expression in R. sphaeroides (Fig. 3A and B). In the initial phase, the expression kinetic of PcrZ is similar to that of pucBA, but the factor of increase is smaller and a steady increase over the time of the experiment was observed (Fig. 3A and B). A constitutive overexpression of PcrZ results in elevated levels of PcrZ, irrespective of the oxygen tension (Fig. 3C and D).6 As a consequence, activation of photosynthesis gene expression is strongly reduced and maximal mRNA levels of pucBA are not reached within 240 min after drop of oxygen tension. It is likely that the steady increase of PcrZ in the R. sphaeroides wild-type helps to counteract excessive photosynthesis gene activation by PrrA. A slight pulse of pucBA expression with a maximum at 120 min is the characteristic feature of this particular incoherent FFL. Overexpression of PcrZ consequently eliminates this characteristic pulse expression. In R. capsulatus, photosynthesis gene expression exhibits an earlier and more precise pulse, although PcrZ is not present. Obviously, this type of FFL is not necessarily required for inhibition of photosynthesis gene activation. The protein-based regulatory system for controlling photosynthesis genes is however not identical in the two species and sRNAs in R. capsulatus have not been identified. Thus, a different factor may take over this inhibiting effect in R. capsulatus.


A mixed incoherent feed-forward loop contributes to the regulation of bacterial photosynthesis genes.

Mank NN, Berghoff BA, Klug G - RNA Biol (2013)

Figure 3. Representative northern blots for pucBA and PcrZ expression kinetics after drop of oxygen tension at time point 0. R. sphaeroides 2.4.1 wild-type (A) and R. sphaeroides PcrZ overexpression strain (pRKPcrZ) (C) pre-cultures were grown overnight under high oxygen conditions (8 mg/L soluble O2) to an OD660nm of 0.6–0.8 (t0). Samples were collected at indicated time points after a shift to low oxygen conditions (0.5 mg/L soluble O2). Fifteen µg of total RNA were separated on 1% agarose gels containing 2.2 M formaldehyde. For pucBA detection, a specific DNA fragment was radioactively labeled with (α-32P)-dCTP using the NEBlot kit (New England Biolabs). A 14S rRNA-specific oligonucleotide was end-labeled with (γ-32P)-ATP and served as loading control. In R. sphaeroides, 23S rRNA is processed to 16S, 14S and 5.8S rRNA fragments by RNase III.46,47 For PcrZ and 5S rRNA, 7.5 µg of total RNA was separated on 10% polyacrylamide gels containing 7 M urea. PcrZ and 5S rRNA-specific oligonucleotides were radioactively end-labeled with (γ-32P)-ATP and were used for detection. 5S rRNA served as loading control. (B) Quantification of northern blot signals for wild-type (A). (D) Quantification of northern blot signals for pRKPcrZ (C). The mean and the standard error are based on three independent biological experiments. The Y axis to the left shows the fold change of pucBA (black squares), the Y axis to the right displays the fold change of PcrZ (black circles).
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Figure 3: Figure 3. Representative northern blots for pucBA and PcrZ expression kinetics after drop of oxygen tension at time point 0. R. sphaeroides 2.4.1 wild-type (A) and R. sphaeroides PcrZ overexpression strain (pRKPcrZ) (C) pre-cultures were grown overnight under high oxygen conditions (8 mg/L soluble O2) to an OD660nm of 0.6–0.8 (t0). Samples were collected at indicated time points after a shift to low oxygen conditions (0.5 mg/L soluble O2). Fifteen µg of total RNA were separated on 1% agarose gels containing 2.2 M formaldehyde. For pucBA detection, a specific DNA fragment was radioactively labeled with (α-32P)-dCTP using the NEBlot kit (New England Biolabs). A 14S rRNA-specific oligonucleotide was end-labeled with (γ-32P)-ATP and served as loading control. In R. sphaeroides, 23S rRNA is processed to 16S, 14S and 5.8S rRNA fragments by RNase III.46,47 For PcrZ and 5S rRNA, 7.5 µg of total RNA was separated on 10% polyacrylamide gels containing 7 M urea. PcrZ and 5S rRNA-specific oligonucleotides were radioactively end-labeled with (γ-32P)-ATP and were used for detection. 5S rRNA served as loading control. (B) Quantification of northern blot signals for wild-type (A). (D) Quantification of northern blot signals for pRKPcrZ (C). The mean and the standard error are based on three independent biological experiments. The Y axis to the left shows the fold change of pucBA (black squares), the Y axis to the right displays the fold change of PcrZ (black circles).
Mentions: Oxygen tension and light are the most important stimuli controlling formation of photosynthetic complexes in R. sphaeroides. A drop of oxygen tension is an activating stimulus and corresponding expression kinetics of photosynthetic complexes were first monitored for R. capsulatus, which shares many factors involved in regulation of photosynthesis genes with R. sphaeroides.23 In R. capsulatus, mRNA levels of photosynthesis genes intensely increased after drop of oxygen tension, reached a maximum after 30–60 min and decreased again. A strong increase of photosynthesis gene expression (e.g., pucBA) is also observable in R. sphaeroides, but maximal expression is reached only 120 min following the drop of oxygen tension. After maximal expression levels are reached, we observed only a slow decrease of expression in R. sphaeroides (Fig. 3A and B). In the initial phase, the expression kinetic of PcrZ is similar to that of pucBA, but the factor of increase is smaller and a steady increase over the time of the experiment was observed (Fig. 3A and B). A constitutive overexpression of PcrZ results in elevated levels of PcrZ, irrespective of the oxygen tension (Fig. 3C and D).6 As a consequence, activation of photosynthesis gene expression is strongly reduced and maximal mRNA levels of pucBA are not reached within 240 min after drop of oxygen tension. It is likely that the steady increase of PcrZ in the R. sphaeroides wild-type helps to counteract excessive photosynthesis gene activation by PrrA. A slight pulse of pucBA expression with a maximum at 120 min is the characteristic feature of this particular incoherent FFL. Overexpression of PcrZ consequently eliminates this characteristic pulse expression. In R. capsulatus, photosynthesis gene expression exhibits an earlier and more precise pulse, although PcrZ is not present. Obviously, this type of FFL is not necessarily required for inhibition of photosynthesis gene activation. The protein-based regulatory system for controlling photosynthesis genes is however not identical in the two species and sRNAs in R. capsulatus have not been identified. Thus, a different factor may take over this inhibiting effect in R. capsulatus.

Bottom Line: Living cells use a variety of regulatory network motifs for accurate gene expression in response to changes in their environment or during differentiation processes.In Rhodobacter sphaeroides, a complex regulatory network controls expression of photosynthesis genes to guarantee optimal energy supply on one hand and to avoid photooxidative stress on the other hand.This point-of-view provides a comparison to other described feed-forward loops and discusses the physiological relevance of PcrZ in more detail.

View Article: PubMed Central - PubMed

Affiliation: Institut für Mikrobiologie und Molekularbiologie; Universität Giessen; Giessen, Germany.

ABSTRACT
Living cells use a variety of regulatory network motifs for accurate gene expression in response to changes in their environment or during differentiation processes. In Rhodobacter sphaeroides, a complex regulatory network controls expression of photosynthesis genes to guarantee optimal energy supply on one hand and to avoid photooxidative stress on the other hand. Recently, we identified a mixed incoherent feed-forward loop comprising the transcription factor PrrA, the sRNA PcrZ and photosynthesis target genes as part of this regulatory network. This point-of-view provides a comparison to other described feed-forward loops and discusses the physiological relevance of PcrZ in more detail.

Show MeSH
Related in: MedlinePlus