PhaF, a Polyhydroxyalkanoate-Granule-Associated Protein of Pseudomonas oleovorans GPo1 Involved in the Regulatory Expression System for pha Genes

Abstract

The phaC1 gene codes for the medium-chain-length polyhydroxyalkanoate (mcl PHA) synthase of Pseudomonas oleovorans GPo1, which produces mcl PHA when grown in an excess of carbon source and under nitrogen limitation. In this work, we have demonstrated, by constructing a recombinant P. oleovoransstrain carrying a phaC1::lacZreporter system, that the phaC1 gene is expressed efficiently in the presence of octanoic acid while its expression is repressed when glucose or citrate is used as the carbon source. Moreover, a P. oleovorans GPo1 mutant (strain GPG-Tc6) expressing higher levels of the reporter gene than the wild-type strain in the presence of glucose or citrate has been generated by mini-Tn5 insertional mutagenesis. Characterization of this mutant allowed us to conclude that phaF, a gene located downstream of the pha gene cluster, was knocked out in this strain. P. oleovorans GPG-Tc6 regained the ability to control phaC1 gene expression when complemented with thephaF wild-type gene. Sequencing data revealed the presence of three complete open reading frames (ORFs) in this region: ORF1 andphaI and phaF genes. The amino acid sequences of the phaI gene product and the N-terminal half of the PhaF protein showed a significant degree of similarity. Furthermore, the primary structure of the PhaF C terminus identifies this protein as a member of the histone H1-like group of proteins. Northern blot analysis showed two transcription units containing phaF, i.e., phaF and phaIF transcripts. Expression of the phaIF operon is more efficient in the presence of octanoic acid and is enhanced by the lack of the PhaF protein. In addition, it has also been demonstrated that both PhaF and PhaI proteins are bound to PHA granules produced by P. oleovorans. A model for the role of PhaF in regulating PHA synthesis is presented.

Regulation of polyhydroxyalkanoate biosynthesis in Pseudomonas putida and Pseudomonas aeruginosa

“…These data suggested a differential regulation of the PHA biosynthesis genes in P. aeruginosa. Furthermore, in P. aeruginosa phaI and phaF are co-transcribed as had been previously suggested for P. oleovorans [9].…”

“…Both PHA synthases have been purified and employed for in vitro synthesis of poly-3-hydroxydecanoate [3,[5][6][7]. The PHA biosynthesis gene locus in P. aeruginosa also includes the phaD (encoding putative transcriptional regulator), phaF and phaI (unknown function) genes, whose homologues in Pseudomonas oleovorans are proposed to be PHA granule-associated structural proteins, whereas PhaF has been also considered as a negative regulator of PHA biosynthesis genes [8,9]. In P. oleovorans under PHA accumulating conditions PhaF is bound to PHA granules, and less PhaF is available for repression, which resulted in enhanced transcription of phaC1 and phaI [9].…”

“…The PHA biosynthesis gene locus in P. aeruginosa also includes the phaD (encoding putative transcriptional regulator), phaF and phaI (unknown function) genes, whose homologues in Pseudomonas oleovorans are proposed to be PHA granule-associated structural proteins, whereas PhaF has been also considered as a negative regulator of PHA biosynthesis genes [8,9]. In P. oleovorans under PHA accumulating conditions PhaF is bound to PHA granules, and less PhaF is available for repression, which resulted in enhanced transcription of phaC1 and phaI [9]. In addition phaC1-lacZ fusions in P. oleovorans indicated repression of phaC1 transcription when gluconate was used, which does not enable PHA accumulation in P. oleovorans.…”

Involvement of poly(3‐hydroxybutyrate) synthesis in catabolite repression of tetralin biodegradation genes in Sphingomonas macrogolitabida strain TFA

“…Alternatively, phasins could still be involved in triggering catabolite repression (Prieto et al, 1999). Transcription of S. macrogolitabida phaP may be independent of phaC but induced by the presence of PHB, as in R. eutropha, where it is known that phasin production responds to production of PHB granules through the transcriptional repressor PhaR (Pötter et al, 2002;York et al, 2002).…”

Smart polyhydroxyalkanoate nanobeads by protein based functionalization

“…PhaF has been shown to have a role as a central player in the machinery, controlling PHA granule segregation and localization in the cell, since it shows a unique ability to bind at least two ligands (the PHA granules and the nucleoid). 7,9,58,92 The peculiar structural organization of PhaF into two domains performing diverse functions (i.e., C-terminal histon-like domain, N-terminal phasin-like domain) supplies an explanation to its biological role. 8,9 Moreover, whether or not P. putida cytoskeletal or other GAP proteins facilitate the organization of granules in needle array like structure (Figure 4), by direct or indirect interaction with PhaF, is still an open question and currently the precise mechanisms by which intermediary PhaF positions the PHA granules are still unknown.…”