CotC-CotU heterodimerization during assembly of the Bacillus subtilis spore coat

Abstract

We report evidence that CotC and CotU, two previously identified components of the Bacillus subtilis spore coat, are produced concurrently in the mother cell chamber of the sporulating cell under the control of sigmaK and GerE and immediately assembled around the forming spore. In the coat, the two proteins interact to form a coat component of 23 kDa. The CotU-CotC interaction was not detected in two heterologous hosts, suggesting that it occurs only in B. subtilis. Monomeric forms of both CotU and CotC failed to be assembled at the surface of the developing spore and accumulated in the mother cell compartment of cells mutant for cotE. In contrast, neither CotU nor CotC accumulated in the mother cell compartment of cells mutant for cotH. These results suggest that CotH is required to protect both CotU and CotC in the mother cell compartment of the sporangium and that CotE is needed to allow their assembly and subsequent interaction at the spore surface.

FIG. 1.

(A) CotC and CotU amino acid alignment. (B) Schematic representation of the cotC-cotU chromosomal region. The arrows and numbers indicate the directions of transcription and the positions on the B. subtilis chromosome, respectively. (C) cotU promoter region. The translational start site (TTG) is in boldface, the transcriptional start site is indicated as +1, and the putative promoter sequences are underlined. The arrow indicates oligonucleotide U-pr-Anti, used for the primer extension experiment shown in Fig. 2B.

FIG. 2.

(A) Expression of a cotU::lacZ transcriptional fusion during sporulation in an otherwise wild-type (open diamonds) or a gerE null mutant (closed squares) strain and of a cotC::lacZ translational fusion in an otherwise wild-type strain (closed diamonds). Background levels of β-galactosidase activity were determined in a wild-type strain bearing no lacZ gene (open squares). Samples were collected at various times after the onset of sporulation. Enzyme activity is expressed in Miller units. The data are the means of three independent experiments. (B) Primer extension analysis of the cotU promoter region performed with total RNA extracted from sporulating cells 8 hours (T8) after the onset of sporulation. Primer extension and sequencing reactions were primed with the synthetic oligonucleotide U-pr-Anti (Table 2).

FIG. 3.

Western blot analysis with anti-CotU antibody of coat proteins extracted from a wild type (wt) and congenic strains with null mutations in cotU, cotC, and cotU cotC. The proteins were fractionated on an 18% polyacrylamide gel and, upon electrotransfer onto nitrocellulose membranes, reacted with CotU-specific rabbit antibodies and then with peroxidase-conjugated secondary antibodies and visualized by the Pierce method. The arrows indicate the apparent molecular weights of the observed proteins. The molecular masses of a marker are also indicated. Identical results were obtained with anti-CotC antibody.

FIG. 4.

Western blot analysis with anti-CotU (A and C) or anti-CotC (B) antibody. Coat proteins were extracted from a wild-type (wt) strain and a congenic strain carrying a cotC::His₆ (C::His₆) or a cotU::His₆ (U::His₆) fusion as indicated. Proteins were fractionated and then blotted (A) or purified through an Ni column and then blotted (B and C). In panels B and C, the wt lane contains unpurified proteins from wild-type spores. The arrows indicate the apparent molecular weights of the observed proteins. Molecular mass markers are also indicated.

FIG. 5.

Western blot analysis with anti-CotC antibody. (A) Proteins extracted from recombinant B. subtilis strains containing the His₆ tag fused at the 5′ (C::His₆ N-term) or 3′ (C::His₆ C-term) end of the cotC gene and from a congenic wild-type strain (wt). The indicated molecular masses refer to the various CotC- and CotU-dependent proteins from a wild-type strain (without the His₆ tag). The arrow indicates the 12-kDa species of CotC. (B) Proteins were extracted from spores of a wild-type B. subtilis strain and of a collection of congenic strains with null mutations in cotA, oxdD, sodA, or tgl. Identical results were obtained with anti-CotU antibody.

FIG. 6.

Western blot of proteins extracted at various times (t₇ to t₁₆) after the onset of sporulation from the forespores of sporulating cells of a wild-type (wt) (A) and a cotC null mutant (B) strain of B. subtilis. The proteins were fractionated on a 15% polyacrylamide gel and, upon electrotransfer onto nitrocellulose membranes, reacted with CotU-specific rabbit antibodies and then with peroxidase-conjugated secondary antibodies and visualized by the Pierce method. The estimated sizes of CotC- and CotU-dependent polypeptides are indicated in kilodaltons.

FIG. 7.

Western blot analysis with anti-CotU antibody. (A) Coat proteins were extracted from a wild-type (wt) and congenic strains with null mutations in cotH or cotE. (B) Proteins were extracted at the indicated times (t₈ to t₁₁) during sporulation from the mother cell compartments of sporulating cultures of a cotE or a cotH mutant strain. The lane labeled “spore” contains coat proteins extracted from spores of a wild-type strain.