Abstract

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The planktonic and biofilm growth mode, the presence of polyhydroxyalkanoate (PHA) granules and the nitrogen availability were considered as parameters to study the regulation of genes involved in PHA biosynthesis in Pseudomonas aeruginosa. The transcriptional start point of the phaIF gene cluster, encoding PHA granule-associated proteins with proposed regulatory function, was experimentally verified.

Gfp and lacZ transcriptional reporter fusions, respectively, were used to analyse promoter activities. In planktonic growth under nitrogen limitation, the phaC promoter (PphaC) showed increased induction, while in the PHA-negative mutant the activity of PphaC was reduced to 25% of the wild type and was independent of nitrogen availability. Promoter activity in biofilms was assessed using 2, 0.05 or 0 g/l of NH4Cl as nitrogen source, respectively. PphaC activity was increased during early biofilm growth, whereas in mature biofilms PphaC activity was preferentially localised to stalks of microcolonies.

Nitrogen starvation led to an increased PphaC activity at the biofilm surface. PHA granule formation was confirmed by electron microscopy in planktonic and in biofilm cells. PphaI activity in planktonic cultures was less dependent on the conditions assayed and presented an oscillatory behaviour. In biofilms, PphaI was strongly activated during early stages of biofilm development, but was inactive in mature biofilms. Under nitrogen starvation PphaI activation resembled that of PphaC.

These data suggested a differential regulation of PHA biosynthesis genes in planktonic and biofilm cells, as well as an important regulatory function of PhaF, when considering nitrogen availability. Interestingly, in biofilms PHA biosynthesis gene regulation showed a spatial distribution similar to rhamnolipid biosynthesis genes.