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Overproduced Brucella abortus PdhS-mCherry forms soluble aggregates in Escherichia coli, partially associating with mobile foci of IbpA-YFP.

Van der Henst C, Charlier C, Deghelt M, Wouters J, Matroule JY, Letesson JJ, De Bolle X - BMC Microbiol. (2010)

Bottom Line: These structures are associated with chaperones like IbpA.Moreover, soluble PdhS-mCherry foci do not systematically colocalize with IpbA-YFP, a marker of inclusion bodies.The dynamic localization of IbpA-YFP foci suggests that the IbpA chaperone could scan the E. coli cell to find its substrates.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Biology Research Unit (URBM), University of Namur (FUNDP), 61 Rue de Bruxelles, 5000 Namur, Belgium.

ABSTRACT

Background: When heterologous recombinant proteins are produced in Escherichia coli, they often precipitate to form insoluble aggregates of unfolded polypeptides called inclusion bodies. These structures are associated with chaperones like IbpA. However, there are reported cases of "non-classical" inclusion bodies in which proteins are soluble, folded and active.

Results: We report that the Brucella abortus PdhS histidine kinase fused to the mCherry fluorescent protein forms intermediate aggregates resembling "non-classical" inclusion bodies when overproduced in E. coli, before forming "classical" inclusion bodies. The intermediate aggregates of PdhS-mCherry are characterized by the solubility of PdhS-mCherry, its ability to specifically recruit known partners fused to YFP, suggesting that PdhS is folded in these conditions, and the quick elimination (in less than 10 min) of these structures when bacterial cells are placed on fresh rich medium. Moreover, soluble PdhS-mCherry foci do not systematically colocalize with IpbA-YFP, a marker of inclusion bodies. Instead, time-lapse experiments show that IbpA-YFP exhibits rapid pole-to-pole shuttling, until it partially colocalizes with PdhS-mCherry aggregates.

Conclusion: The data reported here suggest that, in E. coli, recombinant proteins like PdhS-mCherry may transit through a soluble and folded state, resembling previously reported "non-classical" inclusion bodies, before forming "classical" inclusion bodies. The dynamic localization of IbpA-YFP foci suggests that the IbpA chaperone could scan the E. coli cell to find its substrates.

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Dynamic localization pattern of IbpA-YFP in stationary growth phase E. coli. Fluorescent micrographic images of middle stationary phase bacteria plated on rich medium taken every 2 minutes. A: IbpA-YFP; B: IbpA-YFP (yellow) and PdhS-mCherry (red). C: Fluorescence intensity of IbpA-YFP (green) and PdhS-mCherry (red) fusions at times T0, T0+4 minutes and T0+6 minutes. Scale bar: 1 μm
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Figure 5: Dynamic localization pattern of IbpA-YFP in stationary growth phase E. coli. Fluorescent micrographic images of middle stationary phase bacteria plated on rich medium taken every 2 minutes. A: IbpA-YFP; B: IbpA-YFP (yellow) and PdhS-mCherry (red). C: Fluorescence intensity of IbpA-YFP (green) and PdhS-mCherry (red) fusions at times T0, T0+4 minutes and T0+6 minutes. Scale bar: 1 μm

Mentions: Time-lapse experiments were performed to monitor the kinetics of the cytoplasmic distribution of PdhS-mCherry and IbpA-YFP fusions. Mid stationary growth phase bacteria (t12) were plated on LB agarose pads and observed every two minutes at 37°C (see Materials and Methods). We observed a very dynamic localization pattern of IbpA-YFP foci in bacteria that did not contain a PdhS-mCherry aggregate (Fig. 5A). In contrast, when the PdhS-mCherry aggregate was present in t12 bacteria, IbpA-YFP foci moved from pole to pole until they colocalized with the immobile PdhS-mCherry foci (movie S1, Fig. 5B and 5C), which in turn progressively disappeared, as previously observed (Fig. 2). In the late stationary phase cultures, the large IbpA-YFP polar clusters colocalizing with PdhS-mCherry did not move (data not shown).


Overproduced Brucella abortus PdhS-mCherry forms soluble aggregates in Escherichia coli, partially associating with mobile foci of IbpA-YFP.

Van der Henst C, Charlier C, Deghelt M, Wouters J, Matroule JY, Letesson JJ, De Bolle X - BMC Microbiol. (2010)

Dynamic localization pattern of IbpA-YFP in stationary growth phase E. coli. Fluorescent micrographic images of middle stationary phase bacteria plated on rich medium taken every 2 minutes. A: IbpA-YFP; B: IbpA-YFP (yellow) and PdhS-mCherry (red). C: Fluorescence intensity of IbpA-YFP (green) and PdhS-mCherry (red) fusions at times T0, T0+4 minutes and T0+6 minutes. Scale bar: 1 μm
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2957392&req=5

Figure 5: Dynamic localization pattern of IbpA-YFP in stationary growth phase E. coli. Fluorescent micrographic images of middle stationary phase bacteria plated on rich medium taken every 2 minutes. A: IbpA-YFP; B: IbpA-YFP (yellow) and PdhS-mCherry (red). C: Fluorescence intensity of IbpA-YFP (green) and PdhS-mCherry (red) fusions at times T0, T0+4 minutes and T0+6 minutes. Scale bar: 1 μm
Mentions: Time-lapse experiments were performed to monitor the kinetics of the cytoplasmic distribution of PdhS-mCherry and IbpA-YFP fusions. Mid stationary growth phase bacteria (t12) were plated on LB agarose pads and observed every two minutes at 37°C (see Materials and Methods). We observed a very dynamic localization pattern of IbpA-YFP foci in bacteria that did not contain a PdhS-mCherry aggregate (Fig. 5A). In contrast, when the PdhS-mCherry aggregate was present in t12 bacteria, IbpA-YFP foci moved from pole to pole until they colocalized with the immobile PdhS-mCherry foci (movie S1, Fig. 5B and 5C), which in turn progressively disappeared, as previously observed (Fig. 2). In the late stationary phase cultures, the large IbpA-YFP polar clusters colocalizing with PdhS-mCherry did not move (data not shown).

Bottom Line: These structures are associated with chaperones like IbpA.Moreover, soluble PdhS-mCherry foci do not systematically colocalize with IpbA-YFP, a marker of inclusion bodies.The dynamic localization of IbpA-YFP foci suggests that the IbpA chaperone could scan the E. coli cell to find its substrates.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Biology Research Unit (URBM), University of Namur (FUNDP), 61 Rue de Bruxelles, 5000 Namur, Belgium.

ABSTRACT

Background: When heterologous recombinant proteins are produced in Escherichia coli, they often precipitate to form insoluble aggregates of unfolded polypeptides called inclusion bodies. These structures are associated with chaperones like IbpA. However, there are reported cases of "non-classical" inclusion bodies in which proteins are soluble, folded and active.

Results: We report that the Brucella abortus PdhS histidine kinase fused to the mCherry fluorescent protein forms intermediate aggregates resembling "non-classical" inclusion bodies when overproduced in E. coli, before forming "classical" inclusion bodies. The intermediate aggregates of PdhS-mCherry are characterized by the solubility of PdhS-mCherry, its ability to specifically recruit known partners fused to YFP, suggesting that PdhS is folded in these conditions, and the quick elimination (in less than 10 min) of these structures when bacterial cells are placed on fresh rich medium. Moreover, soluble PdhS-mCherry foci do not systematically colocalize with IpbA-YFP, a marker of inclusion bodies. Instead, time-lapse experiments show that IbpA-YFP exhibits rapid pole-to-pole shuttling, until it partially colocalizes with PdhS-mCherry aggregates.

Conclusion: The data reported here suggest that, in E. coli, recombinant proteins like PdhS-mCherry may transit through a soluble and folded state, resembling previously reported "non-classical" inclusion bodies, before forming "classical" inclusion bodies. The dynamic localization of IbpA-YFP foci suggests that the IbpA chaperone could scan the E. coli cell to find its substrates.

Show MeSH
Related in: MedlinePlus