When soluble extracts were examined by gel permeation combined with fluorescence and Western blot analysis, soluble PdhS-mCherry proteins were identified as a single peak, with a predicted molecular weight between 669 kDa and 20,000 kDa, the upper limit of the fractionation range (Additional file 2, Figure S2). This suggests that the

fusion is able to form multimers with a defined number of monomers, further implying that PdhS-mCherry is folded. Using yeast two-hybrid Selleck Torin 1 assays, it was recently shown that B. abortus PdhS was able to interact with FumC through its amino-terminal domain [18], and with DivK through its carboxy-terminal domain [17]. Interestingly, FumC from Caulobacter MEK162 datasheet crescentus did not interact with B. abortus PdhS [18]. When B. abortus FumC-YFP and DivK-YFP fusions were produced with PdhS-mCherry, colocalization of YFP and mCherry fluorescence signals was observed in mid stationary phase E. coli cells (Fig. 6A, C). Interestingly, both fluorescence signals were overlapping, further suggesting that the shift in fluorescence

signals observed between PdhS-mCherry and IbpA-YFP (Fig. 4) was not an artefact. As a control, we checked that C. crescentus FumC did not colocalize with PdhS-mCherry (Fig. 6B). The ability of PdhS-mCherry to recruit B. abortus DivK-YFP and FumC-YFP but not C. crescentus FumC-YFP suggests that the N-terminal and C-terminal domains of PdhS were at least partially folded. Figure 6 PdhS-mCherry fusion is still able to recruit known partners. PdhS-mCherry localization with (A) B. abortus FumC-YFP, (B) Caulobacter crescentus FumC-YFP, and (C) B. abortus DivK-YFP. Bacteria were cultivated until middle stationary culture phase. Scale bar: 2 μm. All micrographic images were taken with the same magnification. Discussion We report that, when overproduced in E. coli, B. abortus PdhS fused to mCherry

is able to form FAK inhibitor intermediate aggregates of soluble proteins resembling previously reported “”non-classical”" IB [3, 15], before forming “”classical”" IB. These intermediate aggregates ID-8 are very different from “”classical”" IB because they are soluble, are quickly removed when bacteria are placed in rich medium (Fig. 2A), do not systematically colocalize with IbpA-YFP (Fig. 3B) and are still able to recruit known PdhS partners (Fig. 6). The observation of “”intermediate”" aggregates of soluble proteins does not fit with a simple model of IB formation in which unfolded proteins precipitate to form IB immediately after translation. Our observations thus suggest that some proteins could form aggregates of folded and soluble polypeptides before their precipitation into “”classical”" IB.