Niels Krüger

A New Metabolic Link between Fatty Acid de NovoSynthesis and Polyhydroxyalkanoic Acid Synthesis THE PHAG GENE FROM PSEUDOMONAS PUTIDAKT2440 ENCODES A 3-HYDROXYACYL-ACYL CARRIER PROTEIN-COENZYME A TRANSFERASE

To investigate the metabolic link between fatty acid de novo synthesis and polyhydroxyalkanoic acid (PHA) synthesis, we isolated mutants of Pseudomonas putida KT2440 deficient in this metabolic route. The gene phaG was cloned by phenotypic complementation of these mutants; it encoded a protein of 295 amino acids with a molecular mass of 33,876 Da, and the amino acid sequence exhibited 44% amino acid identity to the primary structure of the rhlA gene product, which is involved in the rhamnolipid biosynthesis in Pseudomonas aeruginosa PG201. S1 nuclease protection assay identified the transcriptional start site 239 base pairs upstream of the putative translational start codon. Transcriptional induction of phaG was observed when gluconate was provided, and PHA synthesis occurred from this carbon source. No complementation of the rhlA mutant P. aeruginosa UO299-harboring plasmid pBHR81, expressingphaG gene under lac promoter control, was obtained. Heterologous expression of phaG inPseudomonas oleovorans, which is not capable of PHA synthesis from gluconate, enabled PHA synthesis on gluconate as the carbon source. Native recombinant PhaG was purified by native polyacrylamide gel electrophoresis from P. oleovorans-harboring plasmid pBHR81. It catalyzes the transfer of the acyl moiety from in vitro synthesized 3-hydroxydecanoyl-CoA to acyl carrier protein, indicating that PhaG exhibits a 3-hydroxyacyl-CoA-acyl carrier protein transferase activity.

Isolation of prokaryotic RNA and detection of specific mRNA with biotinylated probes

Abstract A rapid method is presented for the isolation of total RNA of prokaryotic cells and the identification of specific mRNA with high sensitivity using a modified method for the detection of bioytinylated DNA. Total RNA was isolated by a procedure based on the principles of nucleic acid isolation. A significant reduction in mRNA degradation was achieved by (i) a drastic reduction in time for cell separation and (ii) employing a phenol-choloroform-SDS mixture of RNA extraction. RNA was transferred to positively charged nylon membranes and for the first time biotinylated probes were applied for mRNA identification. Genes of Alcaligenes eutrophus of soluble hydrogenase ( hoxS ) and of the ezymes involved in poly(β-hydroybutyrate) formation were used for the establishment of the method.

PHA biosynthesis, its regulation and application of C1-utilizing microorganisms for polyester production

Polyhydroxyalkanoic acids (PHA) represent a storage compound for carbon and/or energy and occur as insoluble inclusions in the cells of a wide range of different bacteria (Steinbuchel 1991) mostly during cultivation of the cells in the presence of an excess of carbon source and if one other essential nutrient limits growth (Anderson, Dawes 1990). In addition to poly(3-hydroxybutyric acid), poly(3HB), which is the most well-known representative of PHA, 90 different hydroxyalkanoic acids have been detected as constituents of PHA from bacterial origin (Steinbuchel, Valentin 1995). These polyesters have been attracted much attention during the last years since they are biodegradable thermoplastics and/or elastomers which exhibit many other interesting properties and which therefore lend themselves for a wide range of different technical applications (Hocking, Marchessault 1994).

Molecular analysis of the Alcaligenes eutrophus PHB-biosynthetic genes: identification of the NH2-terminus of PHB synthase and identification of the transcription start site of phbC

The 12.5-kb EcoRI DNA-fragment PP1 isolated from genomic DNA of Alcaligenes eutrophus H16, which encodes the genes for s-ketothiolase (phbA), NADPH-depedent acetoacetyl-CoA reductase (phbB), and PHB synthase (phbC) has been cloned recently (1). One subclone, which habored the 5.2-kb SmaI-EcoRI subfragment SE52, expressed all three genes and still accumulated PHB up to 50% of its cellular dry weight. SA35, a 3.5-kb SmaI-EcoRI subfragment of SE52, containing the entire structural gene phbC was blunt ended with T4 DNA polymerase and cloned in both orientations into the multicopy vector Bluescript KS-. The resulting hybrid plasmids were referred to as pSAP33 and pSAP37. Nested deletions of both plasmids were generated using the exonuclease III/mung bean nuclease system (Stratagene, San Diego, USA).The deleted plasmids were used for sequence analysis of phbC. In comparison with the DNA sequence data provided of Peoples and Sinskey (2) only one difference, an additional “G” between position “280” and “281”, was observed.