Phosphate concentration regulates transcription of the Acinetobacter polyhydroxyalkanoic acid biosynthetic genes

Abstract

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...

Role of ERAB/l-3-Hydroxyacyl-coenzyme A Dehydrogenase Type II Activity in Aβ-induced Cytotoxicity

“…Its expression was found to be increased in AD brain, and, in vitro, ERAB facilitated A␤ cytotoxicity in neuroblastoma and transfected COS cells. Our initial analysis of the ERAB amino acid sequence showed resemblance to the family of short-chain alcohol dehydrogenases, including hydroxysteroid dehydrogenases, Ke6, and acetoacetyl coenzyme A (CoA) reductases (16). These results suggested a primary role for ERAB in cell metabolism, in addition to having the potential to contribute to A␤-induced cytotoxicity.…”

“…We find that it has the ability to catalyze the oxidation of alcohol groups in a range of substrates, including linear alcohols and estradiol, as well as the reduction of S-acetoacetyl-CoA. To analyze the role of ERAB/HADH II enzymatic activity in potentiation of A␤ toxicity, a catalytically crippled form was prepared containing two substitutions in the highly conserved sequence of residues 168 -172 (YSASK), putatively assigned as part of the active center of the enzyme (16). Cells overexpressing mutant ERAB were relatively protected from A␤ cytotoxicity consequent to overexpression of mutant ␤APP(V717G), as demonstrated by suppression of apoptosis.…”

High Levels of Bioplastic Are Produced in Fertile Transplastomic Tobacco Plants Engineered with a Synthetic Operon for the Production of Polyhydroxybutyrate

“…Genes encoding the Acinetobacter sp. thiolase (phaA) and synthase (phaC; Schembri et al, 1995) and the Bacillus megaterium reductase (phaB; McCool and Cannon, 1999) were chosen from the set of available PHB biosynthetic pathway gene sequences, since their GC content is similar to that of the tobacco plastome and codons with a low frequency of use in the tobacco plastome are either absent or rarely occur. The 5# UTR of gene 10 from bacteriophage T7 (Kuroda and Maliga, 2001) and short (fewer than 56 nucleotides) spacer elements of plastidial origin (Herz et al, 2005) were used upstream of the individual transgenes to optimize expression.…”

“…PHA biosynthetic gene locus (EMBL accession no. L37761; Schembri et al, 1995); rps19/rpl22 spacer, DNA homologous to nucleotides 86,353 to 86,399 of the tobacco plastome, contains an intergenic region between rps19 and rpl22 (Herz et al, 2005); phaA As , gene encoding thiolase from Acinetobacter sp., homologous to nucleotides 4,206 to 5,384 of the Acinetobacter sp. PHA biosynthetic gene locus; 5# UTR rbcL, 15 nucleotides of the 5# leader sequence of the gene encoding the large subunit of Rubisco, homologous to nucleotides 57,580 to 57,594 of the tobacco plastome (Svab and Maliga, 1993); psbD/C spacer, DNA homologous to nucleotides 35,463 to 35,517 of the tobacco plastome, contains the sequence region upstream of the gene encoding PSII 44-kD protein (psbC), which is overlapped by the coding region of the psbD gene encoding the PSII D2 protein (Herz et al, 2005); phaB Bm , gene encoding acetoacetyl-CoA reductase from B. megaterium, homologous to nucleotides 4,758 to 5,501 of the B. megaterium PHA gene cluster (EMBL accession no.…”

“…Genes from Acinetobacter sp. (Schembri et al, 1995) and Bacillus megaterium (McCool and Cannon, 1999) were found to be most similar to the codon usage (i.e. avoids the use of codons with a low frequency of use [less than 10 in 1,000]) and GC content (less than 50%) of the tobacco plastome sequence and were chosen for use in plastid transformation vectors.…”