Generally, prokaryotes store carbon as polyhydroxyalkanoate, starch, or glycogen. The Gram-positive actinomycete Rhodococcus opacus strain PD630 is noteworthy in that it stores carbon in the form of triacylglycerol (TAG). Several studies have demonstrated that R. opacus PD630 can accumulate up to 76% of its cell dry weight as TAG when grown under nitrogen-limiting conditions. While this process is well studied, the underlying molecular and biochemical mechanisms leading to TAG biosynthesis and subsequent storage are poorly understood. We designed a high-throughput genetic screening to identify genes and their products required for TAG biosynthesis and storage in R. opacus PD630. We identified a gene predicted to encode a putative heparin-binding hemagglutinin homolog, which we have termed tadA (triacylglycerol accumulation deficient), as being important for TAG accumulation. Kinetic studies of TAG accumulation in both the wild-type (WT) and mutant strains demonstrated that the tadA mutant accumulates 30 to 40% less TAG than the parental strain (WT). We observed that lipid bodies formed by the mutant strain were of a different size and shape than those of the WT. Characterization of TadA demonstrated that the protein is capable of binding heparin and of agglutinating purified lipid bodies. Finally, we observed that the TadA protein localizes to lipid bodies in R. opacus PD630 both in vivo and in vitro. Based on these data, we hypothesize that the TadA protein acts to aggregate small lipid bodies, found in cells during early stages of lipid storage, into larger lipid bodies and thus plays a key role in lipid body maturation in R. opacus PD630.
