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PhaF, a polyhydroxyalkanoate-granule-associated protein of Pseudomonas oleovorans GPo1 involved in the regulatory
1Institute of Biotechnology, ETH Hönggerberg, CH-8093 Zürich, Switzerland.
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. oleovorans strain carrying a phaC1::lacZ reporter 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 the phaF wild-type gene. Sequencing data revealed the presence of three complete open reading frames (ORFs) in this region: ORF1 and phaI 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.
Insights
Pseudomonas oleovorans GPo1 regulates medium-chain-length polyhydroxyalkanoate (mcl PHA) synthesis via the phaC1 gene. The phaF gene
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Pseudomonas oleovorans GPo1 synthesizes medium-chain-length polyhydroxyalkanoates (mcl PHA) under specific nutrient conditions.
- The phaC1 gene encodes the mcl PHA synthase, crucial for polymer production.
- Understanding the regulation of phaC1 gene expression is key to controlling PHA biosynthesis.
Purpose of the Study:
- To investigate the regulatory mechanisms controlling phaC1 gene expression in P. oleovorans.
- To identify genetic factors involved in the carbon source-dependent regulation of PHA synthesis.
- To elucidate the function of phaF and its role in PHA production.
Main Methods:
- Construction of a phaC1::lacZ reporter system in P. oleovorans.
- Generation of mutants using mini-Tn5 insertional mutagenesis.
- Gene sequencing, complementation studies, and Northern blot analysis.
- Protein binding assays to PHA granules.
Main Results:
- phaC1 gene expression is carbon source-dependent, induced by octanoic acid and repressed by glucose/citrate.
- A mutant (GPG-Tc6) with enhanced phaC1 expression on glucose/citrate was identified, with a knockout in the phaF gene.
- Complementation with wild-type phaF restored normal phaC1 regulation.
- The phaF gene is part of the phaIF operon, and its absence enhances phaIF expression.
- PhaF and PhaI proteins bind to PHA granules.
Conclusions:
- The phaF gene plays a significant role in repressing phaC1 gene expression, particularly under non-inducing carbon sources.
- PhaF is a regulatory protein involved in controlling PHA synthesis in P. oleovorans.
- A regulatory model involving PhaF and PhaI in PHA granule-associated regulation is proposed.