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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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Fluorescent distribution of PdhS-mCherry fusion in stationary growth phase E. coli. A, early stationary phase; B, middle stationary phase; C, late stationary phase. White arrows point to refractile bodies that are only present in the bacteria from the late stationary culture phase. Scale bar: 2 μm. DIC means differential interference contrast (Nomarski). All micrographic images were taken with the same magnification.
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Figure 1: Fluorescent distribution of PdhS-mCherry fusion in stationary growth phase E. coli. A, early stationary phase; B, middle stationary phase; C, late stationary phase. White arrows point to refractile bodies that are only present in the bacteria from the late stationary culture phase. Scale bar: 2 μm. DIC means differential interference contrast (Nomarski). All micrographic images were taken with the same magnification.

Mentions: We used the pCVDH07 plasmid to overexpress the pdhS coding sequence (CDS) fused in frame with the CDS for the fluorescent reporter mCherry (see Materials and Methods). Interestingly, the localization of this fusion in E. coli revealed foci at quarter (2%), mid-cell (10%) and polar (88%) sites of E. coli S17-1 in the stationary phase (n = 200) (Fig. 1B). The PdhS-mCherry was a stable fusion in E. coli, since Western blot analysis using antibodies raised against mCherry revealed a major band with the expected molecular mass for the complete fusion (data not shown). Fusing the pdhS CDS to the yfp or cfp CDS on the same backbone plasmid or overexpressing the pdhS-mCherry fusion in DH10B, TOP10 and MG1655 E. coli strains also generated similar fluorescent foci (data not shown). When a pdhS-mCherry fusion was carried on a low-copy plasmid, there was no polar focus in E. coli, contrary to its expression in B. abortus where PdhS-mCherry monopolar foci were present (data not shown). Other B. abortus proteins (the DivK response regulator, FumA and FumC fumarases) fused to the mCherry N-terminus did not generate fluorescent foci but rather a diffuse signal (data not shown). Taken together, this data suggests that foci formation in E. coli is mainly due to PdhS itself and to the abundance of the whole PdhS-mCherry recombinant protein.


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)

Fluorescent distribution of PdhS-mCherry fusion in stationary growth phase E. coli. A, early stationary phase; B, middle stationary phase; C, late stationary phase. White arrows point to refractile bodies that are only present in the bacteria from the late stationary culture phase. Scale bar: 2 μm. DIC means differential interference contrast (Nomarski). 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 1: Fluorescent distribution of PdhS-mCherry fusion in stationary growth phase E. coli. A, early stationary phase; B, middle stationary phase; C, late stationary phase. White arrows point to refractile bodies that are only present in the bacteria from the late stationary culture phase. Scale bar: 2 μm. DIC means differential interference contrast (Nomarski). All micrographic images were taken with the same magnification.
Mentions: We used the pCVDH07 plasmid to overexpress the pdhS coding sequence (CDS) fused in frame with the CDS for the fluorescent reporter mCherry (see Materials and Methods). Interestingly, the localization of this fusion in E. coli revealed foci at quarter (2%), mid-cell (10%) and polar (88%) sites of E. coli S17-1 in the stationary phase (n = 200) (Fig. 1B). The PdhS-mCherry was a stable fusion in E. coli, since Western blot analysis using antibodies raised against mCherry revealed a major band with the expected molecular mass for the complete fusion (data not shown). Fusing the pdhS CDS to the yfp or cfp CDS on the same backbone plasmid or overexpressing the pdhS-mCherry fusion in DH10B, TOP10 and MG1655 E. coli strains also generated similar fluorescent foci (data not shown). When a pdhS-mCherry fusion was carried on a low-copy plasmid, there was no polar focus in E. coli, contrary to its expression in B. abortus where PdhS-mCherry monopolar foci were present (data not shown). Other B. abortus proteins (the DivK response regulator, FumA and FumC fumarases) fused to the mCherry N-terminus did not generate fluorescent foci but rather a diffuse signal (data not shown). Taken together, this data suggests that foci formation in E. coli is mainly due to PdhS itself and to the abundance of the whole PdhS-mCherry recombinant protein.

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