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Genetic and ultrastructural analysis of different mutants of Pseudomonas putida affected in the poly‐3‐hydroxy‐n‐alkanoate gene cluster
Author(s) -
Sandoval Ángel,
AriasBarrau Elsa,
Arcos Mario,
Naharro Germán,
Olivera Elías R.,
Luengo José M.
Publication year - 2007
Publication title -
environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.954
H-Index - 188
eISSN - 1462-2920
pISSN - 1462-2912
DOI - 10.1111/j.1462-2920.2006.01196.x
Subject(s) - pseudomonas putida , polyhydroxyalkanoates , biology , mutant , plasmid , gene , transformation (genetics) , locus (genetics) , biochemistry , biosynthesis , microbiology and biotechnology , bacteria , genetics
Summary 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 ( PpU Δ pha ). These studies showed that: (i) Pha enzymes cannot be replaced by other proteins in this bacterium, (ii) the transformation of Pp Δ pha with a plasmid containing the locus pha fully restores the synthesis of bioplastics, (iii) the transformation of Pp Δ pha with a plasmid harbouring the gene encoding the polymerase PhaC1 (pMC phaC1 ) permits the synthesis of polyesters (even in absence of pha C2ZDFI); however, in this strain ( Pp UΔ pha ‐pMC pha C1) the number of PHAs granules was higher than in the wild type, (iv) the expression of phaF in PpU Δ pha ‐pMC phaC1 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 Δ fadBA mutant, led to the collection of PHA over‐producer strains.