is capable of synthesizing polyhydroxyalkanoic acids (PHAs) and rhamnolipids, both of which are composed of 3-hydroxydecanoic acids connected by ester bonds, as well as synthesizing the biofilm matrix polymer alginate. In order to study the influence of PHA biosynthesis on rhamnolipid and alginate biosynthesis, as well as stress tolerance and biofilm formation, isogenic knock-out mutants deficient in PHA biosynthesis were generated for PAO1 and the alginate-overproducing FRD1. A gentamicin-resistance cassette was inserted replacing the 3′ region of , the whole of and the 5′ region of . Gas chromatography/mass spectrometry analysis showed that PHA accumulation was completely abolished in both strains. Interestingly, this gene replacement did not abolish rhamnolipid production. Thus, as previously suggested, the PHA synthase is not directly involved in rhamnolipid biosynthesis. In the PHA-negative mutant of mucoid FRD1 alginate biosynthesis was not affected, whereas in the PHA-negative PAO1 mutant an almost threefold increase in biosynthesis was observed compared to the wild-type. Consistently, PHA accumulation in FRD1 contributed only 4·7 % of cell dry weight, which is fourfold less than in PAO1. These data suggest that PHA biosynthesis and alginate biosynthesis are in competition with respect to a common precursor. The surface attachment and biofilm development of the PHA-negative mutants were also compared to those of wild-type strains in glass flow-cell reactors. PHA-negative mutants of PAO1 and FRD1 showed reduced attachment to glass. However, the PAO1 PHA-negative mutant, in contrast to the wild-type, formed a stable biofilm with large, distinct and differentiated microcolonies characteristic of alginate-overproducing strains of . The stress tolerance of PHA-negative mutants with respect to elevated temperature was strongly impaired. These data indicated a functional role for PHA in stress response and tolerance.


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