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Spatial localization of the first and last enzymes effectively connects active metabolic pathways in bacteria.

Meyer P, Cecchi G, Stolovitzky G - BMC Syst Biol (2014)

Bottom Line: We found that out of 857 globular enzymes, at least 219 have a discrete punctuate localization in the cytoplasm and catalyze the first or the last reaction in 60% of biosynthetic pathways.A Gene Ontology analysis of these enzymes reveals an enrichment of terms related to essential metabolic functions in growing cells.We conclude that active biochemical pathways inside the cytoplasm are organized spatially following a rule where their first or their last enzymes localize to effectively connect the different active pathways and thus could reflect the activity state of the cell's metabolic network.

View Article: PubMed Central - PubMed

Affiliation: IBM Computational Biology Center, Yorktown Heights, NY, USA. pmeyerr@us.ibm.com.

ABSTRACT

Background: Although much is understood about the enzymatic cascades that underlie cellular biosynthesis, comparatively little is known about the rules that determine their cellular organization. We performed a detailed analysis of the localization of E.coli GFP-tagged enzymes for cells growing exponentially.

Results: We found that out of 857 globular enzymes, at least 219 have a discrete punctuate localization in the cytoplasm and catalyze the first or the last reaction in 60% of biosynthetic pathways. A graph-theoretic analysis of E.coli's metabolic network shows that localized enzymes, in contrast to non-localized ones, form a tree-like hierarchical structure, have a higher within-group connectivity, and are traversed by a higher number of feed-forward and feedback loops than their non-localized counterparts. A Gene Ontology analysis of these enzymes reveals an enrichment of terms related to essential metabolic functions in growing cells. Given that these findings suggest a distinct metabolic role for localization, we studied the dynamics of cellular localization of the cell wall synthesizing enzymes in B. subtilis and found that enzymes localize during exponential growth but not during stationary growth.

Conclusions: We conclude that active biochemical pathways inside the cytoplasm are organized spatially following a rule where their first or their last enzymes localize to effectively connect the different active pathways and thus could reflect the activity state of the cell's metabolic network.

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Localization dynamics of cell wall synthesis enzymes. A. Red arrow indicates a cell where MurA-CFP (left) and MurG-GFP (middle) colocalize as shown in the overlaid image (right) together with membrane dye (red). B. Model for organization of enzymes from the cell wall synthesis pathway going from a diffuse localization during stationary growth to a discrete concentration of MurA and MurG during exponential growth. C. Left images, Timecourse distribution of enzyme MurA-CFP in B.subtilis during exponential and stationary growth. Right graph, percentage of cells showing over time localized MurA-CFP (blue, right axis) compared to the culture optical density (black, left axis).
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Fig4: Localization dynamics of cell wall synthesis enzymes. A. Red arrow indicates a cell where MurA-CFP (left) and MurG-GFP (middle) colocalize as shown in the overlaid image (right) together with membrane dye (red). B. Model for organization of enzymes from the cell wall synthesis pathway going from a diffuse localization during stationary growth to a discrete concentration of MurA and MurG during exponential growth. C. Left images, Timecourse distribution of enzyme MurA-CFP in B.subtilis during exponential and stationary growth. Right graph, percentage of cells showing over time localized MurA-CFP (blue, right axis) compared to the culture optical density (black, left axis).

Mentions: As the significant list of GO terms reflects enzymatic activities necessary for E.coli cells growing exponentially, we decided to test whether enzyme localization would vary in different growth conditions. We studied the dynamics of cellular organization in exponential and stationary growth in the bacterium B. subtilis. Due to its larger size, B. subtilis has emerged as an excellent model organism for bacterial cytology and in particular to study the very well conserved set of cell wall synthesizing enzymes [25]. We fluorescently tagged the cytoplasmically localized set of enzymes MurABCDEFG (see Additional files 5 and 6: Text S7&S8 Plasmids & Strains) and observed that the fluorescent signal from these fusions showed a diffuse localization in the cytoplasm except for the first enzyme MurA-CFP and the last enzyme MurG-GFP (see Figure 4A). In up to 70% of exponentially growing cells from 10 experiments performed in the strain JDB1925, MurA-CFP forms a bright fluorescent spot per cell (Figure 4A&C), localizing principally at septa (39 ± 5% of at least 100 cells), lateral walls (21 ± 5%), poles (29 ± 5%) and in a few cases cytoplasmically (11 ± 5%). In strain JDB2501 MurG-GFP expressed during exponential growth also localized and in strain JDB2840 cells expressing both MurA-CFP/MurG-GFP show co-localization of both proteins (Figure 4A). This shows that the distribution of the cell wall synthesis enzymes in E.coli as shown in Additional file 1: Figure S1B is conserved in B.subtilis and in both organisms only MurA and MurG show specific localization.Figure 4


Spatial localization of the first and last enzymes effectively connects active metabolic pathways in bacteria.

Meyer P, Cecchi G, Stolovitzky G - BMC Syst Biol (2014)

Localization dynamics of cell wall synthesis enzymes. A. Red arrow indicates a cell where MurA-CFP (left) and MurG-GFP (middle) colocalize as shown in the overlaid image (right) together with membrane dye (red). B. Model for organization of enzymes from the cell wall synthesis pathway going from a diffuse localization during stationary growth to a discrete concentration of MurA and MurG during exponential growth. C. Left images, Timecourse distribution of enzyme MurA-CFP in B.subtilis during exponential and stationary growth. Right graph, percentage of cells showing over time localized MurA-CFP (blue, right axis) compared to the culture optical density (black, left axis).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4279816&req=5

Fig4: Localization dynamics of cell wall synthesis enzymes. A. Red arrow indicates a cell where MurA-CFP (left) and MurG-GFP (middle) colocalize as shown in the overlaid image (right) together with membrane dye (red). B. Model for organization of enzymes from the cell wall synthesis pathway going from a diffuse localization during stationary growth to a discrete concentration of MurA and MurG during exponential growth. C. Left images, Timecourse distribution of enzyme MurA-CFP in B.subtilis during exponential and stationary growth. Right graph, percentage of cells showing over time localized MurA-CFP (blue, right axis) compared to the culture optical density (black, left axis).
Mentions: As the significant list of GO terms reflects enzymatic activities necessary for E.coli cells growing exponentially, we decided to test whether enzyme localization would vary in different growth conditions. We studied the dynamics of cellular organization in exponential and stationary growth in the bacterium B. subtilis. Due to its larger size, B. subtilis has emerged as an excellent model organism for bacterial cytology and in particular to study the very well conserved set of cell wall synthesizing enzymes [25]. We fluorescently tagged the cytoplasmically localized set of enzymes MurABCDEFG (see Additional files 5 and 6: Text S7&S8 Plasmids & Strains) and observed that the fluorescent signal from these fusions showed a diffuse localization in the cytoplasm except for the first enzyme MurA-CFP and the last enzyme MurG-GFP (see Figure 4A). In up to 70% of exponentially growing cells from 10 experiments performed in the strain JDB1925, MurA-CFP forms a bright fluorescent spot per cell (Figure 4A&C), localizing principally at septa (39 ± 5% of at least 100 cells), lateral walls (21 ± 5%), poles (29 ± 5%) and in a few cases cytoplasmically (11 ± 5%). In strain JDB2501 MurG-GFP expressed during exponential growth also localized and in strain JDB2840 cells expressing both MurA-CFP/MurG-GFP show co-localization of both proteins (Figure 4A). This shows that the distribution of the cell wall synthesis enzymes in E.coli as shown in Additional file 1: Figure S1B is conserved in B.subtilis and in both organisms only MurA and MurG show specific localization.Figure 4

Bottom Line: We found that out of 857 globular enzymes, at least 219 have a discrete punctuate localization in the cytoplasm and catalyze the first or the last reaction in 60% of biosynthetic pathways.A Gene Ontology analysis of these enzymes reveals an enrichment of terms related to essential metabolic functions in growing cells.We conclude that active biochemical pathways inside the cytoplasm are organized spatially following a rule where their first or their last enzymes localize to effectively connect the different active pathways and thus could reflect the activity state of the cell's metabolic network.

View Article: PubMed Central - PubMed

Affiliation: IBM Computational Biology Center, Yorktown Heights, NY, USA. pmeyerr@us.ibm.com.

ABSTRACT

Background: Although much is understood about the enzymatic cascades that underlie cellular biosynthesis, comparatively little is known about the rules that determine their cellular organization. We performed a detailed analysis of the localization of E.coli GFP-tagged enzymes for cells growing exponentially.

Results: We found that out of 857 globular enzymes, at least 219 have a discrete punctuate localization in the cytoplasm and catalyze the first or the last reaction in 60% of biosynthetic pathways. A graph-theoretic analysis of E.coli's metabolic network shows that localized enzymes, in contrast to non-localized ones, form a tree-like hierarchical structure, have a higher within-group connectivity, and are traversed by a higher number of feed-forward and feedback loops than their non-localized counterparts. A Gene Ontology analysis of these enzymes reveals an enrichment of terms related to essential metabolic functions in growing cells. Given that these findings suggest a distinct metabolic role for localization, we studied the dynamics of cellular localization of the cell wall synthesizing enzymes in B. subtilis and found that enzymes localize during exponential growth but not during stationary growth.

Conclusions: We conclude that active biochemical pathways inside the cytoplasm are organized spatially following a rule where their first or their last enzymes localize to effectively connect the different active pathways and thus could reflect the activity state of the cell's metabolic network.

Show MeSH
Related in: MedlinePlus