Many prokaryotes are able to accumulate large amounts of lipophilic compounds as inclusion bodies in the cytoplasm. Members of most genera synthesize polymeric lipids such as poly(3-hydroxybutyrate) (PHB) or other polyhydroxyalkanoates (PHAs) (125), whereas accumulation of triacylglycerols (TAGs) and wax esters (WEs) in intracellular lipid-bodies is a property of only a few prokaryotes (10). Like the formation of PHAs, TAG and WE biosynthesis is also promoted in response to stress imposed on the cells and during imbalanced growth, for example by nitrogen limitation, if an abundant carbon source is present at the same time. All these lipids act as storage compounds for energy and carbon needed for maintenance of metabolism and synthesis of cellular metabolites during starvation and in particular if growth resumes. Although neutral lipid metabolism in bacteria, especially WE biosynthesis, has attracted increasing biotechnological interest, there has so far been little interest in medical research in the formation of prokaryotic lipid bodies. However, recent findings suggest that lipid body formation and accumulation of TAGs play an important role in the metabolism of the pathogenic bacteria like Mycobacterium tuberculosis (37,52).In contrast to the restricted occurrence of storage TAGs in prokaryotes, intracellular TAGs are widespread in many eukaryotes (34,35,40,53,65,106,112,132,133). In eukaryotes, storage lipids are also deposited as lipid bodies. Their structure and formation have recently been reviewed in detail by several authors, but additional information on prokaryotic lipid-bodies has been only superficial (98,100,151). In contrast, intracellular accumulation of WEs as a storage lipid is a great exception in eukaryotes and occurs only in jojoba (Simmondsia chinensis) (147), and PHAs are not at all synthesized as a storage compound in eukaryotes.The number of studies investigating the enzymatic and structural fundamentals of storage lipid metabolism in bacteria has increased drastically in the last years, providing the knowledge that storage lipids in bacteria have a similar important metabolic function like in eukaryotes. This review will focus on prokaryotic neutral lipid bodies in comparison with the currently discussed models for lipid body structure and their formation in eukaryotes and with PHA inclusions in prokaryotes.

SummaryNeutral lipid accumulation is frequently observed in some Gram-negative prokaryotes like Acinetobacter sp. and most actinomycetes, including the pathogenic Mycobacterium tuberculosis and antibiotic producing streptomycetes. We examined the formation of wax ester-and triacylglycerol (TAG)-bodies in Acinetobacter calcoaceticus and Rhodococcus opacus using microscopic, immunological and biophysical methods. A general model for prokaryotic lipid-body formation is proposed, clearly differing from the current models for the formation of lipid inclusions in eukaryotes and of poly(hydroxyalkanoic acid) (PHA) inclusions in prokaryotes. Formation of lipid-bodies starts with the docking of wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT) to the cytoplasm membrane. Both, analyses of in vivo and in vitro lipid-body synthesis, demonstrated the formation of small lipid droplets (SLDs), which remain bound to the membraneassociated enzyme. SLDs conglomerated subsequently to membrane-bound lipid-prebodies which are then released into the cytoplasm. The formation of matured lipid-bodies in the cytoplasm occurred by means of coalescence of SLDs inside the lipid prebodies, which are surrounded by a half-unit membrane of phospholipids.

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The triacylglycerol (TAG)-accumulating, hydrocarbon-degrading bacterium Rhodococcus opacus strain PD630 and chemically induced storage-deficient mutants derived from this strain were investigated for their capability to accumulate storage lipids in the cytoplasm during cultivation under nitrogen-limiting conditions. Acylglycerols were analysed by matrix-associated laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) and by reversed-phase HPLC. Fatty acids comprising 13-19 carbon atoms in various acylglycerols constituted up to 76% of the cellular dry weight in gluconate-grown cells, with a significant proportion of odd-numbered fatty acids. Hydrolysis using pancreatic lipase and deacylation with ethyl magnesium bromide were employed to identify the stereospecific distribution of fatty acids at the glycerol. This analysis showed that the fatty acids were not randomly distributed between the three positions of the glycerol backbone. In comparison with common plant fats, where the longer and higher unsaturated fatty acids are predominantly found at position 2, R. opacus PD630 accumulated only the shorter and saturated fatty acids in this position. More than 100 mutants accumulating TAG at a significantly lower rate were obtained by chemical mutagenesis and identified by staining with Sudan Black B. All the mutants showed similar neutral lipid patterns by TLC analysis, with a small distinct spot exhibiting the same R(F) value as TAG; this was identified as a residual amount of TAG by preparative TLC and MALDI-TOF, indicating that this bacterium is possibly capable of synthesizing TAGs by at least two different pathways.

The oleaginous bacterium Rhodococus opacus strain PD630 serves as a model organism to investigate the metabolism of storage triacylglycerols (TAGs) in bacteria. The key enzyme catalysing the last step of TAG biosynthesis in bacteria is a promiscuous acyltransferase (Atf), exhibiting acyl-CoA acyltransferase activity to both diacylglycerols (DGAT activity) and fatty alcohols (wax ester synthase, WS activity). An 800 bp PCR product was obtained from chromosomal DNA of strain PD630 by using degenerate primers designed from conserved stretches of Atf proteins of Acinetobacter baylyi strain ADP1 and Mycobacterium smegmatis mc 2 155. The atf fragment was used as a probe on a strain PD630 gene library, resulting in the identification of a 3948 bp chromosomal DNA fragment containing the complete atf1 gene. An atf1 disruption mutant of strain PD630 exhibited a TAG-leaky phenotype and accumulated up to 50 % less fatty acids than the wild-type, with significantly reduced oleic acid content when cultivated in the presence of gluconate or oleic acid. Whereas DGAT activity was drastically reduced in comparison to the wild-type, WS activity remained almost unchanged in the mutant.RT-PCR analysis of gluconate-grown cells of strain PD630 showed that there is expression of atf1 under conditions of TAG synthesis. To identify additional Atfs in strain PD630, PCR employing non-degenerate primers deduced from Rhodococcus jostii RHA1 sequence data was used. This yielded nine additional atf-homologous genes exhibiting 88-99 % sequence identity to the corresponding strain RHA1 enzymes. Besides Atf1 only Atf2 exhibited high DGAT and/or WS activity when heterologously expressed in Escherichia coli.

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.

Phenyldecane supported growth and lipid accumulation of Rhodococcus opacus PD630 during cultivation under nitrogen-limiting conditions. The results of this study suggested that the hydrocarbon phenyldecane was degraded by monoterminal oxidation, followed by β-oxidation of the alkyl side-chain to phenylacetic acid, and by an additional degradative route for the oxidation of the latter to intermediates of the central metabolism. α-Oxidation of phenyldecanoic acid also occurred to some extent. Phenyldecanoic acid, the monoterminal oxidation product, was also utilized for the biosynthesis of a novel wax ester and novel triacylglycerols. The formation of the wax ester phenyldecylphenyldecanoate probably resulted from the condensation of phenyldecanoic acid and phenyldecanol, which were produced as metabolites during the catabolism of phenyldecane. Two types of triacylglycerol were detected in phenyldecane-grown cells of strain PD630. Triacylglycerols containing only odd-and even-numbered aliphatic fatty acids, as well as triacylglycerols in which one fatty acid was replaced by a phenyldecanoic acid residue, occurred. Other phenyl intermediates, such as phenylacetic acid, phenylpropionic acid, 4-hydroxyphenylpropionic acid, protocatechuate and homogentisic acid, were excreted into the medium during cultivation on phenyldecane. On the basis of the results obtained, pathways for the catabolism and assimilation of phenyldecane by R. opacus PD630 are discussed.

The Ralstonia eutropha H16 phasin PhaP1 is targeted to intracellular triacylglycerol inclusions in Rhodococcus opacus PD630 and Mycobacterium smegmatis mc 2 155, and provides an anchor to target other proteins In Ralstonia eutropha, the H16 phasin PhaP1 represents the major phasin that binds to the surface of polyhydroxyalkanoate (PHA) inclusions. In this study, C-terminal fusions of PhaP1 with enhanced green fluorescent protein (eGFP) and with Escherichia coli b-galactosidase (LacZ) were expressed separately in the triacylglycerol (TAG)-accumulating actinomycetes Rhodococcus opacus PD630 and Mycobacterium smegmatis mc 2 155, employing the M. smegmatis acetamidase (ace) promoter of the Escherichia-Mycobacterium/Rhodococcus shuttle plasmid pJAM2. PhaP1 and the PhaP1 fusion proteins were expressed stably in the recombinant strains. Western blot analysis of cell fractions of Rh. opacus revealed that PhaP1 and the PhaP1-eGFP fusion protein were associated with the TAG inclusions, whereas no phasin or phasin fusion protein was detected in the soluble and membrane fractions. Additional electron microscopy/ immunocytochemistry studies demonstrated that PhaP1 was mainly located on the surface of intracellular TAG inclusions; in addition, some PhaP1 also occurred at the plasma membrane. Fluorescence microscopic investigations of the subcellular distribution of the PhaP1-eGFP fusion protein in vivo and on isolated TAG inclusions revealed that the fusion protein was bound to TAG inclusions at all stages of their formation, and to some extent at the cytoplasmic membrane. The PhaP1-LacZ fusion protein also bound to the TAG inclusions, and could be separated together with the inclusions from Rh. opacus crude extracts, thus demonstrating the immobilization of b-galactosidase activity on the inclusions. This is believed to be the first report demonstrating the ability of PhaP1 to bind to lipid inclusions in addition to PHA inclusions. Furthermore, it was demonstrated that this non-specificity of PhaP1 can be utilized to anchor enzymically active fusion proteins to a matrix of bacterial TAG inclusions.