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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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IbpA-YFP and PdhS-mCherry colocalization pattern in stationary culture phase E. coli. A, Partial colocalization of IbpA-YFP and PdhS-mCherry. Relative fluorescent intensity was computed along the dotted white bar. B, Distribution of relative fluorescent signal as shown in A. In green, fluorescent distribution of IbpA-YFP signal. In red, PdhS-mCherry fluorescent signal.
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Figure 4: IbpA-YFP and PdhS-mCherry colocalization pattern in stationary culture phase E. coli. A, Partial colocalization of IbpA-YFP and PdhS-mCherry. Relative fluorescent intensity was computed along the dotted white bar. B, Distribution of relative fluorescent signal as shown in A. In green, fluorescent distribution of IbpA-YFP signal. In red, PdhS-mCherry fluorescent signal.

Mentions: We transformed the pCVDH07, to overexpress the pdhS-mCherry fusion, in a strain expressing a chromosomal ibpA-yfp fusion, previously used to monitor aggregates in vivo [11]. Using fluorescence microscopy, we observed the PdhS-mCherry aggregates and IbpA-YFP localization in early, mid and late stationary phase bacteria (Fig. 3). During the early stationary phase (t0), the bacteria displayed a diffuse cytoplasmic PdhS-mCherry signal while IbpA-YFP foci were mainly present at the cell poles (Fig. 3A). Surprisingly, in mid stationary phase bacteria (t12), colocalization of PdhS-mCherry with IbpA-YFP was quite rare (Fig. 3B). Indeed, only 15% of these bacteria (n = 250) displayed the two corresponding fluorescent foci at the same poles, 15% at the opposite pole, 15% at an intermediate position (often near midcell) and, in 60% of these bacteria, only one fluorescent focus corresponding to PdhS-mCherry was detectable. Moreover, in the bacteria with both fluorescent signals at the same pole, we systematically observed that PdhS-mCherry and IbpA-YFP did not exactly overlap (Fig. 4). At a later stage of the stationary culture phase (t36), polar colocalization of PdhS-mCherry and IbpA-YFP was frequent (78%) (Fig. 3C), but the signal of both fluorescent fusions was also slightly shifted. In these late stationary phase bacteria, both foci also colocalized with dense refractile bodies seen in differential interference contrast (DIC) (Fig. 3C). At t36, the polar IbpA-YFP foci were more frequent and were larger and brighter compared with non-polar IbpA-YFP foci. Western blot analyses showed that the IbpA-YFP fusion was not cleaved (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)

IbpA-YFP and PdhS-mCherry colocalization pattern in stationary culture phase E. coli. A, Partial colocalization of IbpA-YFP and PdhS-mCherry. Relative fluorescent intensity was computed along the dotted white bar. B, Distribution of relative fluorescent signal as shown in A. In green, fluorescent distribution of IbpA-YFP signal. In red, PdhS-mCherry fluorescent signal.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: IbpA-YFP and PdhS-mCherry colocalization pattern in stationary culture phase E. coli. A, Partial colocalization of IbpA-YFP and PdhS-mCherry. Relative fluorescent intensity was computed along the dotted white bar. B, Distribution of relative fluorescent signal as shown in A. In green, fluorescent distribution of IbpA-YFP signal. In red, PdhS-mCherry fluorescent signal.
Mentions: We transformed the pCVDH07, to overexpress the pdhS-mCherry fusion, in a strain expressing a chromosomal ibpA-yfp fusion, previously used to monitor aggregates in vivo [11]. Using fluorescence microscopy, we observed the PdhS-mCherry aggregates and IbpA-YFP localization in early, mid and late stationary phase bacteria (Fig. 3). During the early stationary phase (t0), the bacteria displayed a diffuse cytoplasmic PdhS-mCherry signal while IbpA-YFP foci were mainly present at the cell poles (Fig. 3A). Surprisingly, in mid stationary phase bacteria (t12), colocalization of PdhS-mCherry with IbpA-YFP was quite rare (Fig. 3B). Indeed, only 15% of these bacteria (n = 250) displayed the two corresponding fluorescent foci at the same poles, 15% at the opposite pole, 15% at an intermediate position (often near midcell) and, in 60% of these bacteria, only one fluorescent focus corresponding to PdhS-mCherry was detectable. Moreover, in the bacteria with both fluorescent signals at the same pole, we systematically observed that PdhS-mCherry and IbpA-YFP did not exactly overlap (Fig. 4). At a later stage of the stationary culture phase (t36), polar colocalization of PdhS-mCherry and IbpA-YFP was frequent (78%) (Fig. 3C), but the signal of both fluorescent fusions was also slightly shifted. In these late stationary phase bacteria, both foci also colocalized with dense refractile bodies seen in differential interference contrast (DIC) (Fig. 3C). At t36, the polar IbpA-YFP foci were more frequent and were larger and brighter compared with non-polar IbpA-YFP foci. Western blot analyses showed that the IbpA-YFP fusion was not cleaved (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