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. 2007 Jul 20;189(19):7062–7068. doi: 10.1128/JB.00770-07

FIG. 2.

FIG. 2.

Complementary subunits affect the copurification of MukB and MukEF with the chromosome. Nucleoids were isolated from MG1655 (MG), OU102 (ΔEF), and GC7528 (ΔB) cells grown in LB medium at 23°C and from arabinose-induced (at 37°C) DH5α cells harboring pBB08 (pEF++). (A) Coomassie-stained 14% polyacrylamide gel analyzing cytoplasmic and nucleoid proteins. The gel resolves 5 μg of cytoplasmic proteins or the scaffold and membrane fractions derived from 5 μg of the nucleoid proteins. BE9F, a mixture of purified MukB and MukEF containing 0.5 μg of MukB-His10, 0.23 μg of MukF, and 0.27 μg of MukE-His9; DNase, 0.5 μg of DNase I. Positions of MukB, MukF, and untagged MukE are indicated with arrowheads. The brightest band among cytoplasmic proteins (apparent molecular mass of 46 kDa) is identified as EF-Tu (43 kDa) from the comparison with the E. coli proteome SWISS-2DPAGE (available via www.expasy.org). The major detected components of the scaffold fraction were marked as RpoB and H-NS from the comparison with previous studies (19). p42 could be the α subunit of RNA polymerase, RpoA (18); the 36.5-kDa RpoA was reported to migrate anomalously slow as a 40-kDa protein in a different gel system (SWISS-2DPAGE). (B and C) Western blot analysis of MukB content in isolated nucleoids. A total of 10 μg of collected fractions (labeled as in Fig. 1) was analyzed along with 30 fmol, 90 fmol, and 300 fmol of purified MukB. (D) Western blot analysis of MukEF copurification with isolated nucleoids. The load was 2.5 μg of fractions from the pEF cells or 5 μg of fractions from the ΔB or ΔEF cells. The lanes with the calibration mixture contain 10 fmol, 40 fmol, and 200 fmol of MukF and 20 fmol, 80 fmol, and 400 fmol of MukE-His9. Note the difference in mobilities of His-tagged (E9) and endogenous (E) MukE. (E) Western blot analysis of MukB content in cells overproducing TopA or the N-terminal fragment of MukB (amino acids 1 to 337) using the same pBAD-based expression system. C, cytoplasmic; S, scaffold; M, membrane; W, whole-cell extract; B, MukB; E, MukE; F, MukF.