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.

In contrast to 16:3 plants like rapeseed (Brassica napus), which contain ␣-linolenic acid (18:3 ⌬9,12,15 ) and hexadecatrienoic acid (16:3 ⌬7,10,13 ) as major polyunsaturated fatty acids in leaves, the silica-less diatom Phaeodactylum tricornutum contains eicosapentaenoic acid (EPA; 20:5 ⌬5,8,11,14,17 ) and a different isomer of hexadecatrienoic acid (16:3 ⌬6,9,12 ). In this report, we describe the characterization of two cDNAs having sequence homology to ⌬12-fatty acid desaturases from higher plants. These cDNAs were shown to code for a microsomal and a plastidial ⌬12-desaturase (PtFAD2 and PtFAD6, respectively) by heterologous expression in yeast (Saccharomyces cerevisiae) and Synechococcus, respectively. Using these systems in the presence of exogenously supplied fatty acids, the substrate specificities of the two desaturases were determined and compared with those of the corresponding rapeseed enzymes (BnFAD2 and BnFAD6). The microsomal desaturases were similarly specific for oleic acid (18:1 ⌬9 ), suggesting that PtFAD2 is involved in the biosynthesis of EPA. In contrast, the plastidial desaturase from the higher plant and the diatom clearly differed. Although the rapeseed plastidial desaturase showed high activity toward the 9-fatty acids 18:1 ⌬9 and 16:1 ⌬7 , in line with the fatty acid composition of rapeseed leaves, the enzyme of P. tricornutum was highly specific for 16:1 ⌬9 . Our results indicate that in contrast to EPA, which is synthesized in the microsomes, the hexadecatrienoic acid isomer found in P. tricornutum (16:3 ⌬6,9,12 ) is of plastidial origin.

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