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

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 (Delta phaP1, Delta phaP2, Delta phaP3, AphaP4, Delta phaP12, Delta phaP123 and Delta phaP1234). When cells were grown under conditions permissive for poly(3HB) accumulation, the wild-type strain and all single-phasin negative mutants (Delta phaP2, Delta phaP3 and AphaP4), with the exception of AphaPl, showed similar growth and poly(3HB) accumulation behaviour, and also the size and number of the granules were identical. The single Delta phaP1 mutant and the Delta phaP12, Delta phaP123 and Delta phaP1234 mutants showed an almost identical growth behaviour; however, they accumulated poly(3HB) at a significantly lower level than wild-type and the single Delta phaP2, Delta phaP3 or Delta phaP4 mutants. Gel-mobility-shift assays and DNasel 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 H 16 was extended and confirmed.