An intracellular poly[D(؊)-3-hydroxybutyrate] (PHB) depolymerase gene (phaZ) has been cloned fromRalstonia eutropha H16 by the shotgun method, sequenced, and characterized. Nucleotide sequence analysis of a 2.3-kbp DNA fragment revealed an open reading frame of 1,260 bp, encoding a protein of 419 amino acids with a predicted molecular mass of 47,316 Da. The crude extract of Escherichia coli containing the PHB depolymerase gene digested artificial amorphous PHB granules and released mainly oligomeric D(؊)-3-hydroxybutyrate, with some monomer. The gene product did not hydrolyze crystalline PHB or freeze-dried artificial amorphous PHB granules. The deduced amino acid sequence lacked sequence corresponding to a classical lipase box, Gly-X-Ser-X-Gly. The gene product was expressed in R. eutropha cells concomitant with the synthesis of PHB and localized in PHB granules. Although a mutant of R. eutropha whose phaZ gene was disrupted showed a higher PHB content compared to the wild type in a nutrient-rich medium, it accumulated PHB as much as the wild type did in a nitrogen-free, carbon-rich medium. These results indicate that the cloned phaZ gene encodes an intracellular PHB depolymerase in R. eutropha., is a storage material produced by some bacteria in response to environmental stress. The PHB biosynthesis genes from such bacteria have been cloned in Escherichia coli and studied in detail (13,24,26). However, only a few studies on the intracellular degradation of PHB have been published. In an in vitro system consisting of native PHB granules from Bacillus megaterium and the soluble fraction from PHB-depleted cells of Rhodospirillum rubrum, the existence of a thermostable activator and a thermolabile depolymerase has been reported (16). Various chemical and physical treatments inactivate the native PHB granules. In intracellular degradation of PHB in Ralstonia eutropha, weak hydrolysis activity against [ 14 C]PHB granules independent of the harvest time of the cells was reported (5). We have detected intracellular PHB depolymerase activity in Zoogloea ramigera I-16-M (18) and R. eutropha H16 (21), using proteasetreated native PHB granules as a substrate.A few intracellular poly-3-hydroxyoctanoate (PHO) depolymerase genes have been cloned. Huisman et al. have cloned an intracellular PHO depolymerase gene from Pseudomonas oleovorans using a PHO degradation mutant that cannot degrade PHO (8). Timm and Steinbüchel have cloned a PHO depolymerase gene from Pseudomonas aeruginosa PAO1 by hybridization using information on the DNA sequence of P. oleovorans (29). In both cases, the gene products have yet to be characterized. Although many extracellular PHB depolymerase genes have been cloned (11), no intracellular PHB depolymerase gene has been cloned to date. We tried unsuccessfully to clone the intracellular PHB depolymerase gene (phaZ) in R. eutropha by Southern hybridization using an extracellular PHB depolymerase gene as a probe. Therefore, we performed shotgun gene cloning by assaying enzyme activity of clones expressed...

Generalized lichen nitidus is a rare disease, and there are only a few reports associating it with a genetic disorder. Here we report a case of generalized lichen nitidus in Russell-Silver syndrome.