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Influence of homologous phasins (PhaP) on PHA accumulation and regulation of their expression by the transcriptional repressor PhaR in Ralstonia eutropha H16

Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstraße 3, 48149 Münster, Germany

Correspondence
Alexander Steinbüchel
steinbu{at}uni-muenster.de

Phasins play an important role in the formation of poly(3-hydroxybutyrate) [poly(3HB)] granules and affect their size. Recently, three homologues of the phasin protein PhaP1 were identified in Ralstonia eutropha strain H16. The functions of PhaP2, PhaP3 and PhaP4 were examined by analysis of R. eutropha H16 deletion strains (phaP1, phaP2, phaP3, phaP4, phaP12, phaP123 and phaP1234). When cells were grown under conditions permissive for poly(3HB) accumulation, the wild-type strain and all single-phasin negative mutants (phaP2, phaP3 and phaP4), with the exception of phaP1, showed similar growth and poly(3HB) accumulation behaviour, and also the size and number of the granules were identical. The single phaP1 mutant and the phaP12, phaP123 and phaP1234 mutants showed an almost identical growth behaviour; however, they accumulated poly(3HB) at a significantly lower level than wild-type and the single phaP2, phaP3 or phaP4 mutants. Gel-mobility-shift assays and DNaseI footprinting experiments demonstrated the capability of the transcriptional repressor PhaR to bind to a DNA region +36 to +46 bp downstream of the phaP3 start codon. The protected sequence exhibited high similarity to the binding sites of PhaR upstream of phaP1, which were identified recently. In contrast, PhaR did not bind to the upstream or intergenic regions of phaP2 and phaP4, thus indicating that the expression of these two phasins is regulated in a different way. Our current model for the regulation of phasins in R. eutropha strain H16 was extended and confirmed.

A list of oligonucleotides used in this study is given in Supplementary Table S1, the appearance of confluently grown colonies of the various Ralstonia eutropha mutants in comparison to the wild-type is shown in Supplementary Fig. S1, and the results of gel-mobility-shift assays of PhaR binding to DNA fragments comprising up- and downstream regions of phaP2, phaP3 and phaP4, and footprinting are shown in Supplementary Fig. S2 with the online version of this paper at http://mic.sgmjournals.org.

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