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Global analysis of photosynthesis transcriptional regulatory networks.

Imam S, Noguera DR, Donohue TJ - PLoS Genet. (2014)

Bottom Line: PrrA regulates ∼34 operons encoding mainly photosynthesis and electron transport functions, while CrpK, a previously uncharacterized Crp-family protein, regulates genes involved in photosynthesis and maintenance of iron homeostasis.Furthermore, CrpK and FnrL share similar DNA binding determinants, possibly explaining our observation of the ability of CrpK to partially compensate for the growth defects of a ΔFnrL mutant.We show that the Rrf2 family protein, MppG, plays an important role in photopigment biosynthesis, as part of an incoherent feed-forward loop with PrrA.

View Article: PubMed Central - PubMed

Affiliation: Program in Cellular and Molecular Biology, University of Wisconsin - Madison, Madison, Wisconsin, United States of America; Department of Bacteriology, University of Wisconsin - Madison, Wisconsin Energy Institute, Madison, Wisconsin, United States of America; DOE Great Lakes Bioenergy Research Center, University of Wisconsin - Madison, Madison, Wisconsin, United States of America.

ABSTRACT
Photosynthesis is a crucial biological process that depends on the interplay of many components. This work analyzed the gene targets for 4 transcription factors: FnrL, PrrA, CrpK and MppG (RSP_2888), which are known or predicted to control photosynthesis in Rhodobacter sphaeroides. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identified 52 operons under direct control of FnrL, illustrating its regulatory role in photosynthesis, iron homeostasis, nitrogen metabolism and regulation of sRNA synthesis. Using global gene expression analysis combined with ChIP-seq, we mapped the regulons of PrrA, CrpK and MppG. PrrA regulates ∼34 operons encoding mainly photosynthesis and electron transport functions, while CrpK, a previously uncharacterized Crp-family protein, regulates genes involved in photosynthesis and maintenance of iron homeostasis. Furthermore, CrpK and FnrL share similar DNA binding determinants, possibly explaining our observation of the ability of CrpK to partially compensate for the growth defects of a ΔFnrL mutant. We show that the Rrf2 family protein, MppG, plays an important role in photopigment biosynthesis, as part of an incoherent feed-forward loop with PrrA. Our results reveal a previously unrealized, high degree of combinatorial regulation of photosynthetic genes and significant cross-talk between their transcriptional regulators, while illustrating previously unidentified links between photosynthesis and the maintenance of iron homeostasis.

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Analysis of the FnrL regulon in R. sphaeroides.(A) A total of 62 FnrL binding sites were identified by ChIP-seq across the R. sphaeroides genome. Binding sites across chromosome 1 are highlighted. MochiView [90] was used for visualization of binding profile. (B) Heat map depicts the differentially expressed FnrL target operons between wild type (WT) and ΔfnrL cells grown on acetate-based media. For brevity only the first members of the target operons are presented. The relative expression of nuoA, bchE and hemA (highlighted in red), which are known to be positively regulated by FnrL, are either not differentially expressed or differentially expressed in the opposite direction. (C) Venn diagram depicting the overlap between our FnrL ChIP-seq analysis and prediction from the large-scale reconstruction of R. sphaeroides transcriptional network. (D) Position weight matrix logo generated for FnrL using targets identified by ChIP-seq.
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pgen-1004837-g001: Analysis of the FnrL regulon in R. sphaeroides.(A) A total of 62 FnrL binding sites were identified by ChIP-seq across the R. sphaeroides genome. Binding sites across chromosome 1 are highlighted. MochiView [90] was used for visualization of binding profile. (B) Heat map depicts the differentially expressed FnrL target operons between wild type (WT) and ΔfnrL cells grown on acetate-based media. For brevity only the first members of the target operons are presented. The relative expression of nuoA, bchE and hemA (highlighted in red), which are known to be positively regulated by FnrL, are either not differentially expressed or differentially expressed in the opposite direction. (C) Venn diagram depicting the overlap between our FnrL ChIP-seq analysis and prediction from the large-scale reconstruction of R. sphaeroides transcriptional network. (D) Position weight matrix logo generated for FnrL using targets identified by ChIP-seq.

Mentions: We determined the genome-wide FnrL binding sites using ChIP-seq with wild type (WT) cells grown under anoxygenic photosynthetic conditions. We reproducibly identified a total of 62 FnrL binding sites across 3 independent ChIP-seq experiments, corresponding to 52 known or predicted operons (S1 Table). These included several sites immediately upstream of genes involved in bacteriochlorophyll synthesis (bchEJGP), early steps in tetrapyrrole biosynthesis (hemN, hemZ and hemA), as well as genes that regulate anaerobic respiration using dimethyl sulfoxide (DMSO) as a terminal electron acceptor (dorS) (Fig. 1A, S1 Table). FnrL binding sites were also found upstream of genes encoding functions for iron transporter (feoABC) and iron sulfur cluster assembly (RSP_1949). When we compared this set of ChIP-seq identified FnrL binding sites to data from ChIP-chip analysis [24], 24 of the 27 FnrL binding sites identified previously were also detected in our analysis (S1 Table). The three previously identified FnrL binding sites not identified in our ChIP-seq analysis do not appear to contain a significant FnrL motif and likely represent false binding events. Furthermore, we found an additional 38 FnrL binding sites in the ChIP-seq dataset, implicating this TF as a direct regulator of a wide variety of new functions, ranging from protein synthesis and substrate transport to polyphosphate dependent phosphorylation and nitrogen metabolism (S1 Table, see Discussion). The larger number of sites identified in the ChIP-seq analysis compared to the previous ChIP-chip analysis for FnrL could be a reflection of the higher sensitivity and improved resolution obtainable with ChIP-seq analysis [30]. For instance, the higher signal to noise ratio of ChIP-seq could potentially allow for identification of relatively weak binding sites, which may be difficult to identify by ChIP-chip. Consistent with this, only 1 of the 24 ChIP-chip identified sites had lower than a 20-fold enrichment in the ChIP-seq dataset (S1 Table). On the other hand, some of these differences could also be the result of differences in peak calling algorithms and thresholds used to identify significant binding sites in the individual datasets.


Global analysis of photosynthesis transcriptional regulatory networks.

Imam S, Noguera DR, Donohue TJ - PLoS Genet. (2014)

Analysis of the FnrL regulon in R. sphaeroides.(A) A total of 62 FnrL binding sites were identified by ChIP-seq across the R. sphaeroides genome. Binding sites across chromosome 1 are highlighted. MochiView [90] was used for visualization of binding profile. (B) Heat map depicts the differentially expressed FnrL target operons between wild type (WT) and ΔfnrL cells grown on acetate-based media. For brevity only the first members of the target operons are presented. The relative expression of nuoA, bchE and hemA (highlighted in red), which are known to be positively regulated by FnrL, are either not differentially expressed or differentially expressed in the opposite direction. (C) Venn diagram depicting the overlap between our FnrL ChIP-seq analysis and prediction from the large-scale reconstruction of R. sphaeroides transcriptional network. (D) Position weight matrix logo generated for FnrL using targets identified by ChIP-seq.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4263372&req=5

pgen-1004837-g001: Analysis of the FnrL regulon in R. sphaeroides.(A) A total of 62 FnrL binding sites were identified by ChIP-seq across the R. sphaeroides genome. Binding sites across chromosome 1 are highlighted. MochiView [90] was used for visualization of binding profile. (B) Heat map depicts the differentially expressed FnrL target operons between wild type (WT) and ΔfnrL cells grown on acetate-based media. For brevity only the first members of the target operons are presented. The relative expression of nuoA, bchE and hemA (highlighted in red), which are known to be positively regulated by FnrL, are either not differentially expressed or differentially expressed in the opposite direction. (C) Venn diagram depicting the overlap between our FnrL ChIP-seq analysis and prediction from the large-scale reconstruction of R. sphaeroides transcriptional network. (D) Position weight matrix logo generated for FnrL using targets identified by ChIP-seq.
Mentions: We determined the genome-wide FnrL binding sites using ChIP-seq with wild type (WT) cells grown under anoxygenic photosynthetic conditions. We reproducibly identified a total of 62 FnrL binding sites across 3 independent ChIP-seq experiments, corresponding to 52 known or predicted operons (S1 Table). These included several sites immediately upstream of genes involved in bacteriochlorophyll synthesis (bchEJGP), early steps in tetrapyrrole biosynthesis (hemN, hemZ and hemA), as well as genes that regulate anaerobic respiration using dimethyl sulfoxide (DMSO) as a terminal electron acceptor (dorS) (Fig. 1A, S1 Table). FnrL binding sites were also found upstream of genes encoding functions for iron transporter (feoABC) and iron sulfur cluster assembly (RSP_1949). When we compared this set of ChIP-seq identified FnrL binding sites to data from ChIP-chip analysis [24], 24 of the 27 FnrL binding sites identified previously were also detected in our analysis (S1 Table). The three previously identified FnrL binding sites not identified in our ChIP-seq analysis do not appear to contain a significant FnrL motif and likely represent false binding events. Furthermore, we found an additional 38 FnrL binding sites in the ChIP-seq dataset, implicating this TF as a direct regulator of a wide variety of new functions, ranging from protein synthesis and substrate transport to polyphosphate dependent phosphorylation and nitrogen metabolism (S1 Table, see Discussion). The larger number of sites identified in the ChIP-seq analysis compared to the previous ChIP-chip analysis for FnrL could be a reflection of the higher sensitivity and improved resolution obtainable with ChIP-seq analysis [30]. For instance, the higher signal to noise ratio of ChIP-seq could potentially allow for identification of relatively weak binding sites, which may be difficult to identify by ChIP-chip. Consistent with this, only 1 of the 24 ChIP-chip identified sites had lower than a 20-fold enrichment in the ChIP-seq dataset (S1 Table). On the other hand, some of these differences could also be the result of differences in peak calling algorithms and thresholds used to identify significant binding sites in the individual datasets.

Bottom Line: PrrA regulates ∼34 operons encoding mainly photosynthesis and electron transport functions, while CrpK, a previously uncharacterized Crp-family protein, regulates genes involved in photosynthesis and maintenance of iron homeostasis.Furthermore, CrpK and FnrL share similar DNA binding determinants, possibly explaining our observation of the ability of CrpK to partially compensate for the growth defects of a ΔFnrL mutant.We show that the Rrf2 family protein, MppG, plays an important role in photopigment biosynthesis, as part of an incoherent feed-forward loop with PrrA.

View Article: PubMed Central - PubMed

Affiliation: Program in Cellular and Molecular Biology, University of Wisconsin - Madison, Madison, Wisconsin, United States of America; Department of Bacteriology, University of Wisconsin - Madison, Wisconsin Energy Institute, Madison, Wisconsin, United States of America; DOE Great Lakes Bioenergy Research Center, University of Wisconsin - Madison, Madison, Wisconsin, United States of America.

ABSTRACT
Photosynthesis is a crucial biological process that depends on the interplay of many components. This work analyzed the gene targets for 4 transcription factors: FnrL, PrrA, CrpK and MppG (RSP_2888), which are known or predicted to control photosynthesis in Rhodobacter sphaeroides. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identified 52 operons under direct control of FnrL, illustrating its regulatory role in photosynthesis, iron homeostasis, nitrogen metabolism and regulation of sRNA synthesis. Using global gene expression analysis combined with ChIP-seq, we mapped the regulons of PrrA, CrpK and MppG. PrrA regulates ∼34 operons encoding mainly photosynthesis and electron transport functions, while CrpK, a previously uncharacterized Crp-family protein, regulates genes involved in photosynthesis and maintenance of iron homeostasis. Furthermore, CrpK and FnrL share similar DNA binding determinants, possibly explaining our observation of the ability of CrpK to partially compensate for the growth defects of a ΔFnrL mutant. We show that the Rrf2 family protein, MppG, plays an important role in photopigment biosynthesis, as part of an incoherent feed-forward loop with PrrA. Our results reveal a previously unrealized, high degree of combinatorial regulation of photosynthetic genes and significant cross-talk between their transcriptional regulators, while illustrating previously unidentified links between photosynthesis and the maintenance of iron homeostasis.

Show MeSH
Related in: MedlinePlus