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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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Related in: MedlinePlus

Analysis of the CrpK regulon in R. sphaeroides.(A) CrpK binding sites across chromosome 1. (B) Comparison of motifs generated for all CrpK and FnrL ChIP-seq identified targets, as well as those for targets exclusive to the CrpK and FnrL ChIP-seq datasets. (C) Pair-wise comparison of the global transcript level data between a ΔfnrL strain and a ΔfnrL strain over-expressing CrpK (ΔfnrL+pIND5-crpk) for all ChIP-seq identified CrpK target operons. Only the first members of the operons are shown. Significantly differentially expressed genes are indicated with *. (D) Fold increase in β-galactosidase activity observed after inducing either CrpK or FnrL synthesis using promoter-lacZ fusions of the bchE, RSP_0697 (Usp), ccoN, RSP_3341 and RSP_2349 promoters, integrated into the chromosome of a ΔfnrL-ΔcrpK reporter strain. Fold inductions represent fold change over the measured basal β-galactosidase activity prior to induction of CrpK or FnrL. (E) Percentage of β-galactosidase activity at mutant bchE promoters relative to the WT promoter. The average growth rate of cultures with FnrL induced was 3.4±0.2 hrs, while that of cultures with CrpK induced was 3.1±0.2 hrs. The error bars in (D) and (E) represent standard error of mean of 3 independent biological replicates.
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pgen-1004837-g003: Analysis of the CrpK regulon in R. sphaeroides.(A) CrpK binding sites across chromosome 1. (B) Comparison of motifs generated for all CrpK and FnrL ChIP-seq identified targets, as well as those for targets exclusive to the CrpK and FnrL ChIP-seq datasets. (C) Pair-wise comparison of the global transcript level data between a ΔfnrL strain and a ΔfnrL strain over-expressing CrpK (ΔfnrL+pIND5-crpk) for all ChIP-seq identified CrpK target operons. Only the first members of the operons are shown. Significantly differentially expressed genes are indicated with *. (D) Fold increase in β-galactosidase activity observed after inducing either CrpK or FnrL synthesis using promoter-lacZ fusions of the bchE, RSP_0697 (Usp), ccoN, RSP_3341 and RSP_2349 promoters, integrated into the chromosome of a ΔfnrL-ΔcrpK reporter strain. Fold inductions represent fold change over the measured basal β-galactosidase activity prior to induction of CrpK or FnrL. (E) Percentage of β-galactosidase activity at mutant bchE promoters relative to the WT promoter. The average growth rate of cultures with FnrL induced was 3.4±0.2 hrs, while that of cultures with CrpK induced was 3.1±0.2 hrs. The error bars in (D) and (E) represent standard error of mean of 3 independent biological replicates.

Mentions: We conducted ChIP-seq analysis using a 3X myc tagged variant of CrpK, which was able to restore photosynthetic growth to a ΔfnrL deletion strain (ΔfnrL+pIND5-crpK-3Xmyc) (S3A Figure) confirming its functionality (the observed difference in growth rate between the strains complemented with a tagged or untagged CrpK protein may reflect alterations in activity or abundance of the individual proteins). We identified a total of 38 binding sites for CrpK in the R. sphaeroides genome (Table 3, Fig. 3A). Consistent with its predicted involvement in regulation of photosynthetic genes, CrpK was found to bind to the upstream regulatory regions of bchEJGP and hemA, both known to be involved in the biosynthesis of photopigments or their tetrapyrrole precursors [35]–[37]. CrpK binding sites were also found upstream of genes encoding iron transporters (feoABC, ccmD) and iron-sulfur cluster binding proteins (rdxBHIS). In addition, 23 (60.5%) of the identified CrpK sites were also identified as FnrL target sites (Table 3, S1 Table, S4 Figure), possibly providing an explanation for the ability of CrpK to at least partially compensate for the loss of FnrL (S3A Figure). The remaining 15 CrpK binding sites were not occupied by FnrL under the conditions we tested (Table 3). On the other hand, FnrL was found bound to 39 sites that were not recognized by CrpK. These observations of TF occupancy for a subset of these overlapping and distinct sites were also verified via independent ChIP-qPCR analysis (S3D Figure), but it should be noted that many of the “CrpK unique sites” (i.e., those not also bound by FnrL) are relatively low enrichment sites in ChIP-seq assays (S4 Figure). However, this set of “CrpK unique sites” all possessed a similar shared motif to other identified CrpK sites, so we consider it likely that these are actually direct targets for control by this TF. The ChIP-seq peaks for CrpK and FnrL at sites bound by both TFs were centered at the same location for both TFs (S4 Figure) and consequently the predicted binding motifs for both TFs bear strong DNA sequence similarity at both shared and unique sites (Fig. 3B). This observation is consistent with general motif type recognized by Crp/Fnr-family TFs and the relatively high degree of amino acid sequence similarity in the predicted DNA binding motifs of CrpK and FnrL [24]. However, subtle differences between the motifs that are assembled by analysis of the “FnrL and CrpK unique sites” could be discerned, which might allow for future computational or experimental discrimination between target sites for each TF (Fig. 3B).


Global analysis of photosynthesis transcriptional regulatory networks.

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

Analysis of the CrpK regulon in R. sphaeroides.(A) CrpK binding sites across chromosome 1. (B) Comparison of motifs generated for all CrpK and FnrL ChIP-seq identified targets, as well as those for targets exclusive to the CrpK and FnrL ChIP-seq datasets. (C) Pair-wise comparison of the global transcript level data between a ΔfnrL strain and a ΔfnrL strain over-expressing CrpK (ΔfnrL+pIND5-crpk) for all ChIP-seq identified CrpK target operons. Only the first members of the operons are shown. Significantly differentially expressed genes are indicated with *. (D) Fold increase in β-galactosidase activity observed after inducing either CrpK or FnrL synthesis using promoter-lacZ fusions of the bchE, RSP_0697 (Usp), ccoN, RSP_3341 and RSP_2349 promoters, integrated into the chromosome of a ΔfnrL-ΔcrpK reporter strain. Fold inductions represent fold change over the measured basal β-galactosidase activity prior to induction of CrpK or FnrL. (E) Percentage of β-galactosidase activity at mutant bchE promoters relative to the WT promoter. The average growth rate of cultures with FnrL induced was 3.4±0.2 hrs, while that of cultures with CrpK induced was 3.1±0.2 hrs. The error bars in (D) and (E) represent standard error of mean of 3 independent biological replicates.
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pgen-1004837-g003: Analysis of the CrpK regulon in R. sphaeroides.(A) CrpK binding sites across chromosome 1. (B) Comparison of motifs generated for all CrpK and FnrL ChIP-seq identified targets, as well as those for targets exclusive to the CrpK and FnrL ChIP-seq datasets. (C) Pair-wise comparison of the global transcript level data between a ΔfnrL strain and a ΔfnrL strain over-expressing CrpK (ΔfnrL+pIND5-crpk) for all ChIP-seq identified CrpK target operons. Only the first members of the operons are shown. Significantly differentially expressed genes are indicated with *. (D) Fold increase in β-galactosidase activity observed after inducing either CrpK or FnrL synthesis using promoter-lacZ fusions of the bchE, RSP_0697 (Usp), ccoN, RSP_3341 and RSP_2349 promoters, integrated into the chromosome of a ΔfnrL-ΔcrpK reporter strain. Fold inductions represent fold change over the measured basal β-galactosidase activity prior to induction of CrpK or FnrL. (E) Percentage of β-galactosidase activity at mutant bchE promoters relative to the WT promoter. The average growth rate of cultures with FnrL induced was 3.4±0.2 hrs, while that of cultures with CrpK induced was 3.1±0.2 hrs. The error bars in (D) and (E) represent standard error of mean of 3 independent biological replicates.
Mentions: We conducted ChIP-seq analysis using a 3X myc tagged variant of CrpK, which was able to restore photosynthetic growth to a ΔfnrL deletion strain (ΔfnrL+pIND5-crpK-3Xmyc) (S3A Figure) confirming its functionality (the observed difference in growth rate between the strains complemented with a tagged or untagged CrpK protein may reflect alterations in activity or abundance of the individual proteins). We identified a total of 38 binding sites for CrpK in the R. sphaeroides genome (Table 3, Fig. 3A). Consistent with its predicted involvement in regulation of photosynthetic genes, CrpK was found to bind to the upstream regulatory regions of bchEJGP and hemA, both known to be involved in the biosynthesis of photopigments or their tetrapyrrole precursors [35]–[37]. CrpK binding sites were also found upstream of genes encoding iron transporters (feoABC, ccmD) and iron-sulfur cluster binding proteins (rdxBHIS). In addition, 23 (60.5%) of the identified CrpK sites were also identified as FnrL target sites (Table 3, S1 Table, S4 Figure), possibly providing an explanation for the ability of CrpK to at least partially compensate for the loss of FnrL (S3A Figure). The remaining 15 CrpK binding sites were not occupied by FnrL under the conditions we tested (Table 3). On the other hand, FnrL was found bound to 39 sites that were not recognized by CrpK. These observations of TF occupancy for a subset of these overlapping and distinct sites were also verified via independent ChIP-qPCR analysis (S3D Figure), but it should be noted that many of the “CrpK unique sites” (i.e., those not also bound by FnrL) are relatively low enrichment sites in ChIP-seq assays (S4 Figure). However, this set of “CrpK unique sites” all possessed a similar shared motif to other identified CrpK sites, so we consider it likely that these are actually direct targets for control by this TF. The ChIP-seq peaks for CrpK and FnrL at sites bound by both TFs were centered at the same location for both TFs (S4 Figure) and consequently the predicted binding motifs for both TFs bear strong DNA sequence similarity at both shared and unique sites (Fig. 3B). This observation is consistent with general motif type recognized by Crp/Fnr-family TFs and the relatively high degree of amino acid sequence similarity in the predicted DNA binding motifs of CrpK and FnrL [24]. However, subtle differences between the motifs that are assembled by analysis of the “FnrL and CrpK unique sites” could be discerned, which might allow for future computational or experimental discrimination between target sites for each TF (Fig. 3B).

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