Functional analyses of the different proteins involved in the synthesis and accumulation of polyhydroxyalkanoates (PHAs) in P. putida U were performed using a mutant in which the pha locus had been deleted (PpUΔpha). These studies showed that: (i) Pha enzymes cannot be replaced by other proteins in this bacterium, (ii) the transformation of PpΔpha with a plasmid containing the locus pha fully restores the synthesis of bioplastics, (iii) the transformation of PpΔpha with a plasmid harbouring the gene encoding the polymerase PhaC1 (pMCphaC1) permits the synthesis of polyesters (even in absence of phaC2ZDFI); however, in this strain (Pppha-pMCphaC1) the number of PHAs granules was higher than in the wild type, (iv) the expression of phaF in PpUΔpha-pMCphaC1 restores the original phenotype, showing that PhaF is involved in the coalescence of the PHAs granules. Furthermore, the deletion of the phaDFI genes in P. putida U considerably decreases (> 70%) the biosynthesis of PHAs consisting of hydroxyalkanoates with aliphatic constituents, and completely prevents the synthesis of those ones containing aromatic monomers. Additional experiments revealed that the deletion of phaD in P. putida U strongly reduces the synthesis of PHA, this effect being restored by PhaF. Moreover, the overexpression of phaF in P. putida U, or in its ΔfadBA mutant, led to the collection of PHA over-producer strains.