Using electron cryotomography, we show that the Gram-negative sporulating bacterium Acetonema longum synthesizes high-density storage granules at the leading edges of engulfing membranes. The granules appear in the prespore and increase in size and number as engulfment proceeds. Typically, a cluster of 8 to 12 storage granules closely associates with the inner spore membrane and ultimately accounts for ∼7% of the total volume in mature spores. Energy-dispersive X-ray spectroscopy (EDX) analyses show that the granules contain high levels of phosphorus, oxygen, and magnesium and therefore are likely composed of polyphosphate (poly-P). Unlike the Gram-positive Bacilli and Clostridia, A. longum spores retain their outer spore membrane upon germination. To explore the possibility that the granules in A. longum may be involved in this unique process, we imaged purified Bacillus cereus, Bacillus thuringiensis, Bacillus subtilis, and Clostridium sporogenes spores. Even though B. cereus and B. thuringiensis contain the ppk and ppx genes, none of the spores from Gram-positive bacteria had granules. We speculate that poly-P in A. longum may provide either the energy or phosphate metabolites needed for outgrowth while retaining an outer membrane
