Ralstonia eutropha has been considered as a bacterium, incorporating hydroxyalkanoates of less than six carbons only into polyhydroxyalkanoates (PHAs). Cells of the wild type cultivated with sodium ...octanoate as the carbon source in the presence of the fatty acid *b-oxidation inhibitor sodium acrylate synthesized PHAs composed of the medium chain length hydroxyalkanoates (3HAMCL) 3-hydroxyhexanoate (3HHx) and 3-hydroxyoctanoate (3HO) as well as of 3-hydroxybutyrate and 3-hydroxyproprionate as revealed by gas chromatography, 1H NMR spectroscopy, and mass spectroscopy. The characterization of the polymer as a tetrapolymer was confirmed by differential solvent extraction and measurement of melting and glass transition temperature depression in the purified polymer compared to PHB. These data suggested that the R. eutropha PHA synthase is capable of incorporating longer chain substrates than suggested by previous in vitro studies. Furthermore, expression of the class II PHA synthase gene phaC1 from P. aeruginosa in R. eutropha resulted in the accumulation of PHAs consisting of 3HAMCL contributing about 3-5% to cellular dry weight. These PHAs were composed of nearly equal molar fractions of 3HO and 3-hydroxydecanoate (3HD) with traces of 3HHx. These data indicated that 3HAMCL-CoA thioesters were diverted from the fatty acid *b-oxidation pathway towards PHA biosynthesis in recombinant R. eutropha.
Abstract
The potential of the production of polyhydroxyalkanoates (PHA), consisting of medium-chain-length (MCL) hydroxyfatty acids (C5–C14), in recombinant Escherichia coli was investigated. E. coli ...mutants affected in fatty acid degradation and fatty acid de novo synthesis were employed. We established the functional expression of the Pseudomonas aeruginosa PHA synthase gene phaC1. The coding region of phaC1 was subcloned via PCR into vector pBluescript SK−. The resulting plasmid pBHR71 enabled functional expression of phaC1 under lac promoter control and conferred synthesis and accumulation of PHA to various strains of E. coli. PHA synthesis was analysed with respect to the carbon source in various E. coli fad and fab mutants. This study provided evidence that intermediates of the fatty acid β-oxidation can be directed to PHA synthesis and that 3-hydroxydecanoyl-CoA is the main substrate for PHA synthase PhaC1 from P. aeruginosa. The E. coli fadB mutant LS1298 containing plasmid pBHR71 and cultivated in LB medium containing 0.5% (w/v) decanoate revealed the strongest accumulation of PHA contributing to about 21% of the cellular dry weight, which was composed of 2.5 mol% 3-hydroxyhexanoate, 20 mol% 3-hydroxyoctanoate, 72.5 mol% 3-hydroxydecanoate and 5 mol% 3-hydroxydodecanoate.
The aim of this work was to study the immunogenicity of an outer membrane (OM) protein (AlgE; 54 kDa) which is produced solely by mucoid, i.e. alginate-producing strains of P. aeruginosa. The source ...of AlgE used for our study was the mucoid strain CF3/M1 originally isolated from sputum of a cystic fibrosis (CF) patient. The purified non-denatured protein served as antigen to raise polyclonal monospecific anti-AlgE antibodies in rabbits and to assay sera from 41 cystic fibrosis (CF) patients for anti-AlgE antibodies. According to clinical protocols the sputa of 22 CF patients were positive for P. aeruginosa, 18 were negative and one case was unknown. Our ELISA studies showed that high titers of anti-AlgE antibodies (IgG) corresponded well with the infection status of the CF patients. None of 23 control sera derived from healthy volunteers contained significant levels of anti-AlgE antibodies. Thus the ELISA should be considered as a sensitive diagnostic tool for the early detection of mucoid P. aeruginosa infections in CF patients. Furthermore, we suggest to include AlgE in a multicomponent experimental vaccine for potential protection of non-colonized CF patients from colonization with mucoid P. aeruginosa.
The two conformers of a cyclohexa‐2, 4‐dienone with different substituents at C(6) on irradiation are believed to undergo ring opening stereospecifically affording a mixture of two configurationally ...isomeric diene‐ketenes (and descendents thereof)‐ Exceptions are generally found for those dienones with one C and one O substituent or even with two C substituents, if one of them carries a polar group at a site able to interact through space with the ring CO group. In these cases, only one of the two anticipated diene‐ketenes (and descendents thereof) is produced. A thorough investigation of the photochemistry of a series of structurally different cyclohexa‐2, 4‐dienones on analytical as well as on preparative scale extends our mechanistic knowledge of the various routes from diene‐ketenes into a variety of compound classes. Novel compound classes accessible to diene‐ketenes are seven‐membered carbocycles (by intramolecular aldolization of the zwitterion of appropriately substituted, transiently formed diene‐(N, O)‐ketene acetals) and β‐lactams (by Staudinger reaction).
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