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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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Stability of PdhS-mCherry aggregates in E. coli grown until the stationary culture phase. Fluorescent micrographic images taken using TxRed filter to visualize mCherry fluorescence. Pictures were taken using the same parameters, at intervals of 10 and 15 min, as indicated. A, middle stationary phase bacteria on agarose pad supplemented with LB medium; B, middle stationary phase bacteria on agarose pad with PBS; C, late stationary phase on LB medium. Scale bar: 2 μm. All micrographic images were taken with the same magnification.
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Figure 2: Stability of PdhS-mCherry aggregates in E. coli grown until the stationary culture phase. Fluorescent micrographic images taken using TxRed filter to visualize mCherry fluorescence. Pictures were taken using the same parameters, at intervals of 10 and 15 min, as indicated. A, middle stationary phase bacteria on agarose pad supplemented with LB medium; B, middle stationary phase bacteria on agarose pad with PBS; C, late stationary phase on LB medium. Scale bar: 2 μm. All micrographic images were taken with the same magnification.

Mentions: When stationary phase bacteria (at t12) showing polar fluorescent PdhS-mCherry aggregates were placed on an agarose pad made with rich medium (LB), fluorescent structures quickly disappeared (in less than 10 minutes) (Fig. 2A). Interestingly, when bacteria of the same culture were placed on an agarose pad containing phosphate buffered saline (PBS), fluorescent foci were still detectable for 3 hours and even increased in size and intensity with the appearance of dense refractile bodies (Fig. 2B). Fluorescence decrease in rich medium did not result from photobleaching, since fluorescence was still detectable after repeat exposure of bacteria on agarose pads without additional rich medium. The "classical" IB present in late stationary phase bacteria (at t36) were still observable when these bacteria were placed on an agarose pad supplemented with LB rich medium (Fig. 2C) or PBS (data not shown). Together, these data suggest that fluorescent foci observed during the mid stationary phase are reversible and different from those observed during the late stationary phase of culture.


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)

Stability of PdhS-mCherry aggregates in E. coli grown until the stationary culture phase. Fluorescent micrographic images taken using TxRed filter to visualize mCherry fluorescence. Pictures were taken using the same parameters, at intervals of 10 and 15 min, as indicated. A, middle stationary phase bacteria on agarose pad supplemented with LB medium; B, middle stationary phase bacteria on agarose pad with PBS; C, late stationary phase on LB medium. Scale bar: 2 μm. All micrographic images were taken with the same magnification.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Stability of PdhS-mCherry aggregates in E. coli grown until the stationary culture phase. Fluorescent micrographic images taken using TxRed filter to visualize mCherry fluorescence. Pictures were taken using the same parameters, at intervals of 10 and 15 min, as indicated. A, middle stationary phase bacteria on agarose pad supplemented with LB medium; B, middle stationary phase bacteria on agarose pad with PBS; C, late stationary phase on LB medium. Scale bar: 2 μm. All micrographic images were taken with the same magnification.
Mentions: When stationary phase bacteria (at t12) showing polar fluorescent PdhS-mCherry aggregates were placed on an agarose pad made with rich medium (LB), fluorescent structures quickly disappeared (in less than 10 minutes) (Fig. 2A). Interestingly, when bacteria of the same culture were placed on an agarose pad containing phosphate buffered saline (PBS), fluorescent foci were still detectable for 3 hours and even increased in size and intensity with the appearance of dense refractile bodies (Fig. 2B). Fluorescence decrease in rich medium did not result from photobleaching, since fluorescence was still detectable after repeat exposure of bacteria on agarose pads without additional rich medium. The "classical" IB present in late stationary phase bacteria (at t36) were still observable when these bacteria were placed on an agarose pad supplemented with LB rich medium (Fig. 2C) or PBS (data not shown). Together, these data suggest that fluorescent foci observed during the mid stationary phase are reversible and different from those observed during the late stationary phase of culture.

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