Study of the interactions between the key spore coat morphogenetic proteins CotE and SpoVID

The capability of Bacillus subtilis spores to withstand extreme environmental conditions is thought to be conferred especially by their outermost proteinaceous protective layer, called the spore coat. Of the over 70 proteins that form the spore coat, only a small subset of them affect its morphogenesis, they are referred to as morphogenetic proteins. In this study we investigated the interaction between two spore coat morphogenetic proteins SpoVID and CotE. SpoVID is involved in the process of spore surface encirclement by individual coat proteins, these include CotE, which controls the assembly of the outer coat layer. Both proteins were proposed to be recruited to a common protein scaffold, but their direct association has not been previously shown. Here we studied the interactions between CotE and SpoVID in vitro for the first time by using molecule recognition force spectroscopy, which allows the detection of piconewton forces between conjugated biological pairs and also facilitates the investigation of dynamic processes. The most probable CotE–CotE unbinding force was 49.4 ± 0.1 pN at a loading rate of 3.16 × 10³ pN/s while that of SpoVID–CotE was 26.5 ± 0.6 pN at a loading rate of 7.8 × 10² pN/s. We further analyzed the interactions with the bacterial two hybrid system and pull-down experiments, which also indicate that SpoVID interacts directly with CotE. In combination with the previously identified direct contacts among SpoIVA, SpoVID and SafA, our data imply that the physical association of key morphogenetic proteins forms a basic skeleton where other coat proteins could be attached.