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A RubisCO-like protein links SAM metabolism with isoprenoid biosynthesis.

Erb TJ, Evans BS, Cho K, Warlick BP, Sriram J, Wood BM, Imker HJ, Sweedler JV, Tabita FR, Gerlt JA - Nat. Chem. Biol. (2012)

Bottom Line: Functional assignment of uncharacterized proteins is a challenge in the era of large-scale genome sequencing.Here, we combine in extracto NMR, proteomics and transcriptomics with a newly developed (knock-out) metabolomics platform to determine a potential physiological role for a ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO)-like protein from Rhodospirillum rubrum.Our studies unraveled an unexpected link in bacterial central carbon metabolism between S-adenosylmethionine-dependent polyamine metabolism and isoprenoid biosynthesis and also provide an alternative approach to assign enzyme function at the organismic level.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Champaign, IL, USA.

ABSTRACT
Functional assignment of uncharacterized proteins is a challenge in the era of large-scale genome sequencing. Here, we combine in extracto NMR, proteomics and transcriptomics with a newly developed (knock-out) metabolomics platform to determine a potential physiological role for a ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO)-like protein from Rhodospirillum rubrum. Our studies unraveled an unexpected link in bacterial central carbon metabolism between S-adenosylmethionine-dependent polyamine metabolism and isoprenoid biosynthesis and also provide an alternative approach to assign enzyme function at the organismic level.

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Proteome and transcriptome analysis of R. rubrum cells that were grown aerobically with sulfate or MTA as sole sulfur source(A) Differential induced gel electrophoresis (DIGE) analysis of changes in the proteome of R. rubrum. Proteins up-regulated in MTA-grown cells are shown in cyan, proteins up-regulated in sulfate-grown cells are shown in red, whereas proteins that are not changed under both conditions are shown in white. The calculated pI (first dimension) and the molecular mass standards (second dimension) of the DIGE gel are given. For more information on the five proteins highlighted in green that were selected for identification, see Supplementary Table 4. (B) RNA sequencing (RNAseq) analysis of changes in the transcriptome of R. rubrum. Genes annotated on the chromosome of R. rubrum, are represented by dots according to their physical location on the chromosome and their fold change in mRNA-level. Classical housekeeping genes are listed separately and highlighted by light gray dots in the graph. For more information on all transcripts up-regulated more than 30-fold, see Supplementary Table 5. (C) The “thiol cluster” that was identified by both DIGE and RNAseq analysis. The four proteins of the thiol cluster that were identified by DIGE are numbered and highlighted in green. Transcripts of the thiol-cluster that were identified by RNAseq. are highlighted in cyan, and the fold-upregulation of each gene is given separately.
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Figure 6: Proteome and transcriptome analysis of R. rubrum cells that were grown aerobically with sulfate or MTA as sole sulfur source(A) Differential induced gel electrophoresis (DIGE) analysis of changes in the proteome of R. rubrum. Proteins up-regulated in MTA-grown cells are shown in cyan, proteins up-regulated in sulfate-grown cells are shown in red, whereas proteins that are not changed under both conditions are shown in white. The calculated pI (first dimension) and the molecular mass standards (second dimension) of the DIGE gel are given. For more information on the five proteins highlighted in green that were selected for identification, see Supplementary Table 4. (B) RNA sequencing (RNAseq) analysis of changes in the transcriptome of R. rubrum. Genes annotated on the chromosome of R. rubrum, are represented by dots according to their physical location on the chromosome and their fold change in mRNA-level. Classical housekeeping genes are listed separately and highlighted by light gray dots in the graph. For more information on all transcripts up-regulated more than 30-fold, see Supplementary Table 5. (C) The “thiol cluster” that was identified by both DIGE and RNAseq analysis. The four proteins of the thiol cluster that were identified by DIGE are numbered and highlighted in green. Transcripts of the thiol-cluster that were identified by RNAseq. are highlighted in cyan, and the fold-upregulation of each gene is given separately.

Mentions: Finally, to identify reactions and enzymes following the release of methanethiol, we compared the proteomes of MTA- and sulfate-grown cells. Five proteins were identified that were highly up-regulated during growth on MTA as sole sulfur source (Supplementary Table 4); among these were two putative O-acetyl-homoserine sulfhydrylases (Rru_A0774, Rru_A0784). Notably, four of these five proteins (Rru_A0779, Rru_A0792, and the two sulfhydrylases) were encoded by the same genetic region of fourteen different genes presumably involved in thiol metabolism at the level of cysteine and methionine (“thiol cluster”, Fig. 6). A complementary transcriptome analysis using mRNA sequencing confirmed the results of the proteomic study and allowed a more detailed view on the differences between MTA- and sulfate-grown cells. Whereas the relative transcript levels of the majority (>99%) of the 3933 annotated genes of R. rubrum remained essentially unchanged for both conditions, including the genes involved in methanethiol release in agreement with their housekeeping function, the relative levels of transcripts for all fourteen genes of the “thiol cluster” were increased between 30- and 320-fold in MTA-grown cells (Supplementary Table 5, Fig. 6). Although this expression pattern resembled a generalized stress response by sulfur deprivation, we realized that expression of the “thiol cluster” and its putative O-acetyl-L-homoserine sulfhydrylases would provide R. rubrum with the metabolic capability to recapture methanethiol in the form of methionine, as reported recently21,22. Indeed, biochemical characterization of heterologously expressed Rru_A0774 confirmed the enzyme as methanethiol-specific O-acetyl-L-homoserine sulfhydrylase (Supplementary Table 3). This closed the gap between methanethiol release and methionine formation, explaining how R. rubrum recycles methanethiol when grown with MTA as sole sulfur source (Fig. 4).


A RubisCO-like protein links SAM metabolism with isoprenoid biosynthesis.

Erb TJ, Evans BS, Cho K, Warlick BP, Sriram J, Wood BM, Imker HJ, Sweedler JV, Tabita FR, Gerlt JA - Nat. Chem. Biol. (2012)

Proteome and transcriptome analysis of R. rubrum cells that were grown aerobically with sulfate or MTA as sole sulfur source(A) Differential induced gel electrophoresis (DIGE) analysis of changes in the proteome of R. rubrum. Proteins up-regulated in MTA-grown cells are shown in cyan, proteins up-regulated in sulfate-grown cells are shown in red, whereas proteins that are not changed under both conditions are shown in white. The calculated pI (first dimension) and the molecular mass standards (second dimension) of the DIGE gel are given. For more information on the five proteins highlighted in green that were selected for identification, see Supplementary Table 4. (B) RNA sequencing (RNAseq) analysis of changes in the transcriptome of R. rubrum. Genes annotated on the chromosome of R. rubrum, are represented by dots according to their physical location on the chromosome and their fold change in mRNA-level. Classical housekeeping genes are listed separately and highlighted by light gray dots in the graph. For more information on all transcripts up-regulated more than 30-fold, see Supplementary Table 5. (C) The “thiol cluster” that was identified by both DIGE and RNAseq analysis. The four proteins of the thiol cluster that were identified by DIGE are numbered and highlighted in green. Transcripts of the thiol-cluster that were identified by RNAseq. are highlighted in cyan, and the fold-upregulation of each gene is given separately.
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Related In: Results  -  Collection

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Figure 6: Proteome and transcriptome analysis of R. rubrum cells that were grown aerobically with sulfate or MTA as sole sulfur source(A) Differential induced gel electrophoresis (DIGE) analysis of changes in the proteome of R. rubrum. Proteins up-regulated in MTA-grown cells are shown in cyan, proteins up-regulated in sulfate-grown cells are shown in red, whereas proteins that are not changed under both conditions are shown in white. The calculated pI (first dimension) and the molecular mass standards (second dimension) of the DIGE gel are given. For more information on the five proteins highlighted in green that were selected for identification, see Supplementary Table 4. (B) RNA sequencing (RNAseq) analysis of changes in the transcriptome of R. rubrum. Genes annotated on the chromosome of R. rubrum, are represented by dots according to their physical location on the chromosome and their fold change in mRNA-level. Classical housekeeping genes are listed separately and highlighted by light gray dots in the graph. For more information on all transcripts up-regulated more than 30-fold, see Supplementary Table 5. (C) The “thiol cluster” that was identified by both DIGE and RNAseq analysis. The four proteins of the thiol cluster that were identified by DIGE are numbered and highlighted in green. Transcripts of the thiol-cluster that were identified by RNAseq. are highlighted in cyan, and the fold-upregulation of each gene is given separately.
Mentions: Finally, to identify reactions and enzymes following the release of methanethiol, we compared the proteomes of MTA- and sulfate-grown cells. Five proteins were identified that were highly up-regulated during growth on MTA as sole sulfur source (Supplementary Table 4); among these were two putative O-acetyl-homoserine sulfhydrylases (Rru_A0774, Rru_A0784). Notably, four of these five proteins (Rru_A0779, Rru_A0792, and the two sulfhydrylases) were encoded by the same genetic region of fourteen different genes presumably involved in thiol metabolism at the level of cysteine and methionine (“thiol cluster”, Fig. 6). A complementary transcriptome analysis using mRNA sequencing confirmed the results of the proteomic study and allowed a more detailed view on the differences between MTA- and sulfate-grown cells. Whereas the relative transcript levels of the majority (>99%) of the 3933 annotated genes of R. rubrum remained essentially unchanged for both conditions, including the genes involved in methanethiol release in agreement with their housekeeping function, the relative levels of transcripts for all fourteen genes of the “thiol cluster” were increased between 30- and 320-fold in MTA-grown cells (Supplementary Table 5, Fig. 6). Although this expression pattern resembled a generalized stress response by sulfur deprivation, we realized that expression of the “thiol cluster” and its putative O-acetyl-L-homoserine sulfhydrylases would provide R. rubrum with the metabolic capability to recapture methanethiol in the form of methionine, as reported recently21,22. Indeed, biochemical characterization of heterologously expressed Rru_A0774 confirmed the enzyme as methanethiol-specific O-acetyl-L-homoserine sulfhydrylase (Supplementary Table 3). This closed the gap between methanethiol release and methionine formation, explaining how R. rubrum recycles methanethiol when grown with MTA as sole sulfur source (Fig. 4).

Bottom Line: Functional assignment of uncharacterized proteins is a challenge in the era of large-scale genome sequencing.Here, we combine in extracto NMR, proteomics and transcriptomics with a newly developed (knock-out) metabolomics platform to determine a potential physiological role for a ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO)-like protein from Rhodospirillum rubrum.Our studies unraveled an unexpected link in bacterial central carbon metabolism between S-adenosylmethionine-dependent polyamine metabolism and isoprenoid biosynthesis and also provide an alternative approach to assign enzyme function at the organismic level.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Champaign, IL, USA.

ABSTRACT
Functional assignment of uncharacterized proteins is a challenge in the era of large-scale genome sequencing. Here, we combine in extracto NMR, proteomics and transcriptomics with a newly developed (knock-out) metabolomics platform to determine a potential physiological role for a ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO)-like protein from Rhodospirillum rubrum. Our studies unraveled an unexpected link in bacterial central carbon metabolism between S-adenosylmethionine-dependent polyamine metabolism and isoprenoid biosynthesis and also provide an alternative approach to assign enzyme function at the organismic level.

Show MeSH
Related in: MedlinePlus