The polyhydroxyalkanoic acid (PHA) biosynthetic gene locus was cloned and characterized from an Acinetobacter sp. isolated from activated sludge. Nucleotide sequence analysis identified three clustered genes, phaA Ac (encoding a -ketothiolase), phaB Ac (encoding an acetoacetyl coenzyme A reductase), and phaC Ac (encoding a PHA synthase). In addition, an open reading frame (ORF1) with potential to encode a 13-kDa protein was identified within this locus. The sequence of the putative translational product of ORF1 does not show significant similarity to any sequences in the database. A plasmid containing the Acinetobacter pha locus conferred the ability to accumulate poly--hydroxybutyrate on its Escherichia coli host. These genes appear to lie in an operon transcribed by two promoters upstream of phaB Ac , an apparent constitutive promoter, and a second promoter induced by phosphate starvation and under pho regulon control. These as well as a number of additional potential transcription start points were identified by a combination of primer extension and promoter-chloramphenicol acetyltransferase gene fusion studies carried out in Acinetobacter or E. coli transformants.Polyhydroxyalkanoic acids (PHAs) are a family of bacterial polyesters synthesized by a wide range of organisms under conditions of nutrient limitation in the presence of an excess carbon and energy source (1). These polymers have attracted significant attention for their potential commercial exploitation as biodegradable plastics (22). The most abundant and best-studied PHA is poly--hydroxybutyrate (PHB), a linear, unbranched homopolymer built up of (R)-3-hydroxybutyric acid units.The physiology and molecular genetics of PHB biosynthesis have been extensively studied in Alcaligenes eutrophus (33). More recently, genes involved in PHA metabolism have also been cloned from numerous other organisms (see the review in reference 32). In most organisms, PHB is synthesized via a three-step pathway which involves the condensation of two molecules of acetyl coenzyme A (acetyl-CoA) to acetoacetylCoA via a -ketothiolase (PhaA), reduction of acetoacetylCoA to D-(Ϫ)--hydroxybutyryl-CoA via an NADPH-dependent acetoacetyl-CoA reductase (PhaB), and polymerization to PHB via a PHA synthase (PhaC) (33).Recently, the PHA synthase gene was cloned from an Acinetobacter strain isolated from a modified activated sludge treatment plant (31). Acinetobacter species have been suggested to be the major organisms responsible for enhanced biological phosphate removal from wastewater treated in alternating anaerobic/aerobic activated sludge systems (5). Biochemical models of this overall process involve the breakdown of polyphosphate and synthesis of PHB during the anaerobic stage and breakdown of PHB and production of polyphosphate in the subsequent aerobic stage (8). Strains of Acinetobacter which can accumulate up to 10% phosphorus (10) and 15% PHB (28) on a dry weight basis have been isolated.In A. eutrophus, all three PHA biosynthetic enzymes are synthesized constitutiv...