Preparative isolation of lipid inclusions from Rhodococcus opacus and Rhodococcus ruber and identification of granule-associated proteins

Abstract

Triacylglycerol granules synthesized and accumulated by Rhodococcus opacus and Rhodococcus ruber were isolated by glycerol density gradient centrifugation. Whereas only one type of granule could be isolated from R. opacus, two types of granules with different specific densities were isolated from R. ruber. Both types of R. ruber granules showed a similar content of triacylglycerols and poly(3-hydroxybutyrate- co-3-hydroxyvalerate), but the protein profiles of both types were significantly different. The granules with the lower specific density were colorless; the granules with the higher specific density had a deep orange pigmentation. Solubilization studies revealed three different groups of granule-associated proteins: (1) unspecifically bound proteins, (2) relatively weakly associated proteins, and (3) proteins that resisted solubilization by treatment with 2 M NaCl, 2% (w/v) Triton X-114, 6 M guanidinium hydrochloride, up to 8% (w/v) SDS, and proteolytic digestion. The strong association of proteins of the last group suggested that these may play a specific role in the synthesis or mobilization of storage lipids or in the structure of the granules. The N-terminal amino acid sequences of the most tightly bound proteins were obtained. Proteins of low molecular weight with striking sequence similarity to the ribosomal protein L7 from various actinomycetes were always copurified with the granules.

Identification of the major functional proteins of prokaryotic lipid droplets

“…Similar studies involving the isolation and proteomic analysis of lipid-containing granules/lipid inclusions from Rhodococcus opacus PD630 and Rhodococcus ruber have previously been carried out ( 42 ). When the lipid inclusions of these two bacteria were isolated, they were also found to contain a large number of proteins.…”

The proteomics of lipid droplets: structure, dynamics, and functions of the organelle conserved from bacteria to humans

“…However, it was not until recent proteomic, biochemical, and molecular studies in bacteria that this conservation across species was demonstrated to extend into the prokaryotic kingdom. The earliest of the studies on bacterial lipid droplets was conducted with Rhodococcus ruber ( 42 ). The isolated fraction was defi ned as "lipid inclusions."…”

Nonorganellar Acyl Carrier Protein from Oleaginous Yeast Is a Homologue of Ribosomal Protein P2

“…It was observed that another ribosomal protein L7 was found to be copurified with the lipid inclusion bodies obtained from oleaginous bacteria like Rhodococcus opacus PD360 and Rhodococcus ruber. The significance of this association is not clear (32).…”

“…The cDNA library construction kit was from Stratagene (La Jolla, CA). [1-14 C]Palmitoyl-CoA (51 mCi/mmol), [1-14 C]oleic acid (55 mCi/mmol), and [␣- 32 P]dATP/dCTP (30 Ci/mmol) were obtained from PerkinElmer Life Sciences. Restriction enzymes, deoxynucleotidyl terminal transferase, Pfu polymerase, low melting agarose, deoxynucleotidyl triphosphates, Sephadex G-10, G-50, EDTA, isopropyl-␤-D-thiogalactopyranoside (IPTG), 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside (X-gal), and all other molecular biology reagents were from Amersham Biosciences.…”