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Applied and Environmental Microbiology, August 2004, p. 4440-4448, Vol. 70, No. 8
0099-2240/04/$08.00+0 DOI: 10.1128/AEM.70.8.4440-4448.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Polyhydroxyalkanoate (PHA) Accumulation in Sulfate-Reducing Bacteria and Identification of a Class III PHA Synthase (PhaEC) in Desulfococcus multivorans
Tran Hai,1 Daniela Lange,2 Ralf Rabus,2 and Alexander Steinbüchel1*Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, D-48149 Münster,1 Max-Planck-Institut für Marine Mikrobiologie, D-28359 Bremen, Germany2
Received 30 November 2003/ Accepted 14 April 2004
Seven strains of sulfate-reducing bacteria (SRB) were tested for the accumulation of polyhydroxyalkanoates (PHAs). During growth with benzoate Desulfonema magnum accumulated large amounts of poly(3-hydroxybutyrate) [poly(3HB)]. Desulfosarcina variabilis (during growth with benzoate), Desulfobotulus sapovorans (during growth with caproate), and Desulfobacterium autotrophicum (during growth with caproate) accumulated poly(3HB) that accounted for 20 to 43% of cell dry matter. Desulfobotulus sapovorans and Desulfobacterium autotrophicum also synthesized copolyesters consisting of 3-hydroxybutyrate and 3-hydroxyvalerate when valerate was used as the growth substrate. Desulfovibrio vulgaris and Desulfotalea psychrophila were the only SRB tested in which PHAs were not detected. When total DNA isolated from Desulfococcus multivorans and specific primers deduced from highly conserved regions of known PHA synthases (PhaC) were used, a PCR product homologous to the central region of class III PHA synthases was obtained. The complete pha locus of Desulfococcus multivorans was subsequently obtained by inverse PCR, and it contained adjacent phaEDm and phaCDm genes. PhaCDm and PhaEDm were composed of 371 and 306 amino acid residues and showed up to 49 or 23% amino acid identity to the corresponding subunits of other class III PHA synthases. Constructs of phaCDm alone (pBBRMCS-2::phaCDm) and of phaEDmCDm (pBBRMCS-2::phaEDmCDm) in various vectors were obtained and transferred to several strains of Escherichia coli, as well as to the PHA-negative mutants PHB–4 and GPp104 of Ralstonia eutropha and Pseudomonas putida, respectively. In cells of the recombinant strains harboring phaEDmCDm small but significant amounts (up to 1.7% of cell dry matter) of poly(3HB) and of PHA synthase activity (up to 1.5 U/mg protein) were detected. This indicated that the cloned genes encode functionally active proteins. Hybrid synthases consisting of PhaCDm and PhaE of Thiococcus pfennigii or Synechocystis sp. strain PCC 6308 were also constructed and were shown to be functionally active.
* Corresponding author. Mailing address: Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstrasse 3, D-48149 Münster, Germany. Phone: 49-251-8339821. Fax: 49-251-8338388. E-mail: steinbu{at}uni-muenster.de.
Applied and Environmental Microbiology, August 2004, p. 4440-4448, Vol. 70, No. 8
0099-2240/04/$08.00+0 DOI: 10.1128/AEM.70.8.4440-4448.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
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