CotE binds to CotC and CotU and mediates their interaction during spore coat formation in Bacillus subtilis

Abstract

CotE is a morphogenic protein that controls the assembly of the coat, the proteinaceous structure that surrounds and protects the spore of Bacillus subtilis. CotE has long been thought to interact with several outer coat components, but such interactions were hypothesized from genetic experiment results and have never been directly demonstrated. To study the interaction of CotE with other coat components, we focused our attention on CotC and CotU, two outer coat proteins known to be under CotE control and to form a heterodimer. We report here the results of pull-down experiments that provide the first direct evidence that CotE contacts other coat components. In addition, coexpression experiments demonstrate that CotE is needed and sufficient to allow formation of the CotC-CotU heterodimer in a heterologous host.

FIG. 1.

Western blots of proteins extracted 10 h after the onset of sporulation (t₁₀) from sporulating cells of a spoIVA mutant or a spoIVA cotE double mutant (A) and from the mother cell fraction of a strain overexpressing cotH (indicated as cotH*) (B). Proteins were fractionated on 15% polyacrylamide gels, electrotransferred to membranes, and reacted with anti-CotU antibody. Identical results were obtained with anti-CotC antibody. MW, molecular weights in thousands; wt, wild type.

FIG. 2.

Results of a pull-down experiment performed by binding CotC-His (A) or CotU-His (B) to Ni-NTA magnetic beads. Untagged CotE was then added, and flowthrough (FT), washed (W), and eluted (E1 and E2) proteins collected as described in Materials and Methods. Proteins were fractionated on 12.5% polyacrylamide gels, electrotransferred to membranes, and reacted with anti-CotC, anti-CotU, and anti-CotE antibodies. The same experiment was also performed without CotC-His or CotU-His (C).

FIG. 3.

Coexpression experiment results. Extracts from E. coli cells overproducing CotC and CotU (lane 1), CotC, CotU, and CotE (lane 2) and CotE (lane 3) were fractionated on 15% polyacrylamide gels, electrotransferred to membranes, and reacted with anti-CotC (A) or anti-CotE (B) antibody. Results identical to those of panel A were obtained with anti-CotU antibody (not shown). Panel C shows the position of the three peptides identified as diagnostic of CotC or CotU by the MALDI-TOF experiment results shown in Table 2.