Figure 1.
Production of CotH in a cotG null mutant.
(A) SDS-PAGE fractionation of coat proteins from a wild type strain (PY79) and isogenic strains carrying null mutations in cotG (ER203) or in cotH (ER220). A molecular weight marker is also present and the size of relevant bands indicated. (B) Western blot with anti-CotH antibody of the same three strains analyzed in panel A. The arrow points to the CotH specific band.
Figure 2.
Construction of a single cotG mutant.
(A) Thick gray and black arrows indicate the coding parts of cotG and cotH, respectively. Dashed arrow indicates the mRNA produced from the cotG and cotH promoters, as already reported. Site of insertion of the additional base in the cotG coding sequence (wild type sequence) that causes the formation of a premature stop codon (mutant sequence). Western blot analysis with anti-CotH (B) and anti-CotG (C) antibodies of proteins extracted by SDS treatment from wild type and isogenic mutant spores. The mutants genotype relative to the cotG cotH and amyE loci is indicated. Arrows point the CotH and CotG specific bands.
Figure 3.
Germination efficiency and lysozyme-resistance assays.
Spores derived from wild type (PY79, black circles), cotG null (AZ604, white circles), cotH null (ER220, white squares) and cotGcotH null (AZ603, black squares) were tested for germination efficiency (A) and for lysozime resistance (B). Germination was induced by Asn-GFK and measured as percentage of loss of optical density at 580 nm. Similar results were obtained by using L-Ala to induce germination. A cotE null strain (black triangles) known to be sensitive to lysozyme has been used as positive control during the lysozime treatment. Error bars are based on the standard deviation of 4 independent experiments.
Figure 4.
Western blot analysis of proteins extracted from mature spores of wild type (PY79, lane 1), ΔcotGΔcotH (AZ603, lane 2), ΔcotGΔcotH amyE::cotGcotH (AZ608, lane 3), ΔcotGΔcotH amyE::cotGstopcotH (AZ604, lane 4), cotH::spc (ER220, lane 5) and ΔcotGΔcotH amyE::cotG (AZ607, lane 6 of panel B) strains. For CotA and CotB detection (panel A) the proteins have been extracted by SDS treatment while for CotC and CotU detection (panel B) the NaOH treatment has been used. Proteins (25 µg) were reacted with CotA, CotB and CotC specific rabbit antibodies and then with peroxidase-conjugated secondary antibodies and visualized by the Pierce method. The estimated size of CotB, CotC and CotU is indicated.
Figure 5.
SDS-PAGE and Fluorescence analysis.
(A) Proteins released after treatment with SDS of spores of the indicated strains were fractionated on a 12,5% polyacrilamide gel. The arrow indicates the 41 kDa band correspoding to CotS (18). The gel was stained with Coomassie brilliant blue. (B) Strains carrying the cotS::gfp fusion were analyzed by phase-contrast (PC) and fluorescence (F) microscopy. The bottom panel reports a merge of the two images. Exposure time was 588 ms in all cases.
Figure 6.
CotG and phosphorylation sites.
Results of a mass spectrometry analysis of peptides derived from trypsine digestion of CotG are reported. Unambiguosly identified sites of phosphorylation are indicated. Tripeptides containing a phosphate moiety are underlined; the random coiled tandem repeats region is in red.
Table 1.
Bacillus subtilis strains used in this study.