FORMATION OF INTRACYTOPLASMIC LIPID INCLUSIONS BY RHODOCOCCUS-OPACUS STRAIN-PD630

An oleaginous hydrocarbon-degrading Rhodococcus opacus strain (PD630) was isolated from a soil sample. The cells were able to grow on a vari ety of substrates and to produce large amounts of three different type s of intracellular inclusions during growth on alkanes, phenylalkanes, or non-hydrocarbon substrates. Electron microscopy revealed large num bers of electron-transparent inclusions with a sphere-like structure. In addition, electron-dense inclusions representing polyphosphate and electron-transparent inclusions with an elongated disc-shaped morpholo gy occurred in small amounts. The electron-transparent inclusions of a lkane- or gluconate-grown cells were composed of neutral lipids (98%, w/w), phospholipids (1.2%, w/w), and protein (0.8%, w/w). The major co mponent of the cellular inclusions was triacylglycerols; minor amounts of diacylglycerols and probably also some free fatty acids were also present. Free fatty acids and/or fatty acids in acylglycerols in cells of R. opacus amounted up to 76 or 87% of the cellular dry weight in g luconate- or olive-oil-grown cells, respectively. The fatty acid compo sition of the inclusions depended on the substrate used for cultivatio n. In cells cultivated on n-alkanes, the composition of the fatty acid s was related to the substrate, and intermediates of the P-oxidation p athway, such as hexadecanoic or pentadecanoic acid, were among the acy lglycerols. Hexadecanoic acid was also the major fatty acid (up 36% of total fatty acids) occurring in the lipid inclusions of gluconate-gro wn cells. This indicated that strain PD630 utilized beta-oxidation and de novo fatty acid biosynthesis for the synthesis of storage lipids. Inclusions isolated from phenyldecane-grown cells contained mainly the non-modified substrate and phenylalkanoic acids derived from the hydr ocarbon oxidation, such as phenyldecanoic acid, phenyloctanoic acid, a nd phenylhexanoic acid, and approximately 5% (w/w) of diacylglycerols. The lipid inclusions seemed to have definite structures, probably wit h membranes at their surfaces, which allow them to maintain their shap e, and with some associated proteins, probably involved in the inclusi on formation.