Amino acids in the Bacillus subtilis morphogenetic protein SpoIVA with roles in spore coat and cortex formation

Bacterial spores are protected from the environment by a proteinaceous coat and a layer of specialized peptidoglycan called the cortex. In Bacillus su btilis, the attachment of the coat to the spore surface and the synthesis o f the cortex both depend on the spore protein SpoIVA. To identify functiona lly important amino acids of SpoIVA, we generated and characterized strains bearing random point mutations of spoIVA that result in defects in coat an d cortex formation. One mutant resembles the null mutant, as sporulating ce lls of this strain lack the cortex and the coat forms a swirl in the surrou nding cytoplasm instead of a shell around the spore. We identified a second class of six mutants with a partial defect in spore assembly. In sporulati ng cells of these strains, we frequently observed swirls of mislocalized co at in addition to a coat surrounding the spore, in the same cell. Using imm unofluorescence microscopy, we found that in two of these mutants, SpoIVA f ails to localize to the spore, whereas in the remaining strains, localizati on is largely normal. These mutations identify amino acids involved in targ eting of SpoIVA to the spore and in attachment of the coat. We also isolate d a large set of mutants producing spores that are unable to maintain the d ehydrated state. Analysis of one mutant in this class suggests that spores of this strain accumulate reduced levels of peptidoglycan with an altered s tructure.