To test for the existence of complexes containing three MreB isoforms, we isolated protein complexes from cells expressing, in turn, His-tagged MreB, Mbl and MreBH. MreB–His was expressed from a Pxyl promoter at the amyE locus, to avoid the complication of having the important mreC and mreD genes downstream from the mreB gene (which was deleted in frame). Mbl–His and MreBH–His fusions were expressed from their natural promoters at their native loci. The growth rates and cell shapes of these strains were indistinguishable from that of the wild type (cells expressing MreB–His were cultivated in the presence of 0.25% xylose), indicating that all three fusion proteins are functional (data not shown). The theoretical molecular mass of MreB, Mbl and MreBH is 35.8, 35.7 and 35.5 kDa respectively. Purified proteins from cell cultures without cross-linking treatment were detected as a major single band of just under 40 kDa for MreB and Mbl, and a weak band of just over 40 kDa for MreBH, corresponding to the approximate molecular masses of His-tagged MreB, Mbl and MreBH fusions (Fig. 6A, lanes 1–3). After cross-linking, several co-purified proteins were also detected in these complexes (Fig. 6A, lanes 4–6). The most prominent additional bands were, in each case, slightly higher in mobility than the His-tagged species, likely corresponding to the untagged MreB-family proteins. For the MreB–His and Mbl–His complexes we anticipated that the second bands might correspond to Mbl and MreB respectively. In accordance with this prediction, these bands disappeared when complexes were purified from isogenic strains bearing deletions of the corresponding genes (i.e. mbl and mreB respectively; Fig. 6B, a and b). In both cases liquid chromatography tandem mass spectrometry analysis (LC-MS/MS) confirmed the identities of these band (data not shown). Since MreBH was less abundant in the cells (see Fig. 6A, lane 3), it was hard to detect as a clear visible band in the MreB or Mbl complexes. However, LC-MS/MS analysis detected MreBH in both the MreB–His and Mbl–His complexes, and also demonstrated the presence of MreB and Mbl in the MreBH complex (Fig. 6A, lane 6 and arrows). Furthermore, the intensity of the major band in the Mbl complex was increased when MreBH was overexpressed (Fig. 6B, c, arrow). Therefore, all three MreB isoforms can readily be detected in complex with each other. As a control, cells expressing His-tagged DnaC (a component of the replisome) were analysed in parallel, as shown previously (Kawai et al., 2009). However, no MreB isoforms were detected in equivalent samples from the MreB–His complexes.
Figure 6. Complex association of MreB isoforms. Separation and visualization of protein complexes containing his-tagged MreB, Mbl and MreBH by SDS-PAGE and Colloidal Coomassie staining. MreB, Mbl, MreBH and TufA were identified by LC-MS/MS analysis (arrowheads). TufA is a non-specific background protein (Ishikawa et al., 2006; Kawai et al., 2009). A. Protein complexes containing his-tagged MreB (strain 3738; lanes 1 and 4), Mbl (strain YK403; lanes 2 and 5) and MreBH (strain YK404; lanes 3 and 6) were purified from cultures with (lanes 4–6) or without (lanes 1–3) cross-linking treatment. B. Protein complexes containing MreB–His in the presence or absence of Mbl (a) and Mbl–His in the presence or absence of MreB (b) were separated. Mbl complexes were purified from cultures of strain YK828 (mbl–hisΔmreB amyE::PspacHY–mreBH) grown in the presence or absence of 0.5 mM IPTG (c).
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