Polyhydroxyalkanoate Inclusion Body-Associated Proteins and Coding Region in Bacillus megaterium

Abstract

Polyhydroxyalkanoic acids (PHA) are carbon and energy storage polymers that accumulate in inclusion bodies in many bacteria and archaea in response to environmental conditions. This work presents the results of a study of PHA inclusion body-associated proteins and an analysis of their coding region in Bacillus megaterium 11561. A 7,917-bp fragment of DNA was cloned and shown to carry a 4,104-bp cluster of 5 pha genes,phaP, -Q, -R, -B, and -C. The phaP and -Q genes were shown to be transcribed in one orientation, each from a separate promoter, while immediately upstream, phaR, -B, and -C were divergently transcribed as a tricistronic operon. Transfer of this gene cluster to Escherichia coliand to a PhaC− mutant of Pseudomonas putidagave a Pha+ phenotype in both strains. Translational fusions to the green fluorescent protein localized PhaP and PhaC to the PHA inclusion bodies in living cells. The data presented are consistent with the hypothesis that the extremely hydrophilic protein PhaP is a storage protein and suggests that PHA inclusion bodies are not only a source of carbon, energy, and reducing equivalents but are also a source of amino acids.

PCR cloning of type II polyhydroxyalkanoate biosynthesis genes from two Pseudomonas strains

“…PHA biosynthesis genes have been cloned and expressed, their genetic organization has been well documented and four classes of PHA biosynthesis loci have been characterized [3,8,9]. Type II pha loci have so far been found only in Pseudomonas strains.…”

Co-expression of 3-ketoacyl-ACP reductase and polyhydroxyalkanoate synthase genes induces PHA production in Escherichia coli HB101 strain

“…The deduced amino acid sequence of fabG Ec is homologous to that of NADPH-dependent acetoacetyl-CoA reductase genes (phaB) of Ralstonia eutropha or Zoogloea ramigeria. Recently, a PHA synthesis gene cluster in Bacillus megaterium was cloned [20]. Interestingly, the deduced amino acid sequence of the phaB gene showed high homology (50% identity, 66% similarity) to that of FabG of Synechocystis PCC 6803, suggesting that FabG and PhaB might be derived from the same origin.…”

Effect of over-expression of phasin gene fromAeromonas hydrophilaon biosynthesis of copolyesters of 3-hydroxybutyrate and 3-hydroxyhexanoate

“…First-strand cDNA was synthesized using a reverse transcription PCR kit (SNBC Co., ShangHai, China) in a 20 ll reaction volume containing 8 ll total RNA, 1 ll RNase inhibitor and 2 ll oligo(dT) 18 primer, 4 ll 5 · first-strand buffer, 2 ll dNTP mix, 2 ll DTT and 1 ll MMuLV reverse transcriptase. Quantitative PCR was performed using the GeneAmp 5700 sequence Detection System (PE Biosystems, Foster City, CA, USA).…”

“…Phasin and its structural gene, phaP, were found in various microorganisms. These proteins have been shown to influence the PHA biosynthesis and the size and number of PHA granules [14][15][16][17][18][19]. Recently, three homologous proteins PhaP2, PhaP3 and PhaP4 of the phasin protein PhaP1 were found in Wautersia eutropha (formerly Ralstonia eutropha) [20].…”