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The capability for biosynthesis of neutral lipids is widely distributed in nature and is found in animals and plants as well as microorganisms. In bacteria, the most abundant class of neutral lipids are polyhydroxyalkanoic acids serving as intracellular carbon and energy storage compound (1), but also few examples of substantial triacylglycerol (TAG) 1 accumulation have been reported for species mainly belonging to the actinomycetes genera: Mycobacterium (2), Nocardia, and Rhodococcus (3) and Streptomyces (4). Furthermore, biosynthesis of wax esters (oxoesters of long-chain primary fatty alcohols and longchain fatty acids) has been frequently reported for members of the genus Acinetobacter (5).TAGs are the dominating storage lipid in animals, plants, and eukaryotic microorganisms. TAG biosynthesis is involved in animals in numerous processes such as regulation of plasma TAG concentration, fat storage in adipocytes, and milk production (6). In plants, TAG synthesis is mainly important for the generation of seed oils (7). Using diacylglycerol (DAG) as a substrate, three different classes of enzymes are known that mediate TAG formation (reviewed in Ref. 8). Acyl-CoA:DAG acyltransferase (DGAT) catalyzes the acylation of DAG using acyl-CoA as a substrate. Two DGAT families designated as DGAT1 and DGAT2 are known that exhibit no sequence homologies to each other. Members of the DGAT1 gene family occur in animals and plants (9 -12), whereas members of the DGAT2 gene family were found in animals (13), plants (14), and yeast (15). In human, one DGAT1-related gene and five DGAT2-related genes were identified (13). Recently, DGAT has attracted great interest since it is a potential therapeutical target for obesity treatment (16). Acyl-CoA-independent TAG synthesis is mediated by a phospholipid:DAG acyltransferase found in yeast and plants that uses phospholipids as acyl donors for DAG esterification (17). A third alternative mechanism present in animals and plants is TAG synthesis by a DAG-DAG-transacylase, which uses DAG as acyl donor and acceptor yielding TAG and monoacylglycerol (18,19), although no gene coding such a transacylase could be identified as yet.Wax esters have diverse and important biological functions including coating of aerial surfaces of plants as epicuticular waxes to provide protection against desiccation, ultraviolet light, and attack of pathogens; regulation of buoyant density as the principal component of the spermaceti oil of sperm whales; and serving as energy storage materials in the seeds of the jojoba plant (20). The latter is the main natural source of wax esters for commercial applications since the world-wide ban on whale hunting. However, the high price of jojoba oil has limited its use. Wax esters have a multitude of important technical applications in a variety of areas, including medicine, cosmetics, and food industries as well as their more traditional usage as lubricants. Acinetobacter calcoaceticus accumulates wax esters intracellularly as insoluble inclusions under growth-limiting c...

The oxazine dye Nile blue A and its fluorescent oxazone form, Nile red, were used to develop a simple and highly sensitive staining method to detect poly(3-hydroxybutyric acid) and other polyhydroxyalkanoic acids (PHAs) directly in growing bacterial colonies. In contrast to previously described methods, these dyes were directly included in the medium at concentrations of only 0.5 microgram/ml, and growth of the cells occurred in the presence of the dyes. This allowed an estimation of the presence of PHAs in viable colonies at any time during the growth experiment and a powerful discrimination between PHA-negative and PHA-positive strains. The presence of Nile red or Nile blue A did not affect growth of the bacteria. This viable-colony staining method was in particular applicable to gram-negative bacteria such as Azotobacter vinelandii, Escherichia coli, Pseudomonas putida, and Ralstonia eutropha. It was less suitable for discriminating between PHA-negative and PHA-positive strains of gram-positive bacteria such as Bacillus megaterium or Rhodococcus ruber, but it could also be used to discriminate between wax-ester- and triacylglycerol-negative and -positive strains of Acinetobacter calcoaceticus or Rhodococcus opacus. The potential of this new method and its application to further investigations of PHA synthases and PHA biosynthesis pathways are discussed.

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An overview is provided on the diversity of biosynthetic polyhydroxyalkanoic acids, and all hitherto known constituents of these microbial storage compounds are listed. The occurrence of 91 different hydroxyalkanoic acids reflects the low substrate specificity of polyhydroxyalkanoic acid synthases which are the key enzymes of polyhydroxyalkanoic acid biosynthesis. In addition, the importance of bacterial anabolism and catabolism, which provide the coenzyme A thioesters of the respective hydroxyalkanoic acids as substrates to these PHA synthases, is emphasized.