Available online at www.sciencedirect.com Journal of Biotechnology 133 (2008) 442–452 The polyhydroxyalkanoate biosynthesis genes are differentially regulated in planktonic- and biofilm-grown Pseudomonas aeruginosa Andrea Campisano, Joerg Overhage, Bernd H.A. Rehm ∗ Institute of Molecular Biosciences, Massey University, Private Bag 11222, Palmerston North, New Zealand Received 24 April 2007; received in revised form 31 October 2007; accepted 12 November 2007 Abstract 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 NH 4 Cl 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. © 2007 Elsevier B.V. All rights reserved. Keywords: Pseudomonas aeruginosa; PHA; Polyhydroxyalkanoate; Biofilms 1. Introduction Many bacteria are capable of synthesizing intracellular poly- hydroxyalkanoate (PHA) granules as carbon storage material (Rehm and Steinbuchel, 1999). PHA has also been considered as electron sink under anaerobic conditions to enable recycling of reducing equivalents. Recently it was shown that PHA accumu- lation impacts on Pseudomonas aeruginosa biofilm architecture (Pham et al., 2004). P. aeruginosa contains two functional PHA synthases belonging to class II, which are the key enzymes in PHA biosynthesis (Qi et al., 2000; Rehm, 2003). P. aerugi- nosa PAO1 synthesizes PHA by diverting intermediates of fatty acid metabolism employing the specific linking enzymes enoyl- CoA hydratase (PhaJ) and transacylase (PhaG) (Hoffmann et al., 2002; Rehm et al., 1998; Tsuge et al., 2000). ∗ Corresponding author. Tel.: +64 6 350 5515x7890; fax: +64 6 350 5688. E-mail address: B.Rehm@massey.ac.nz (B.H.A. Rehm). The PHA biosynthesis gene cluster of P. aeruginosa com- prises four genes (Fig. 1A) encoding two PHA synthases, the intracellular depolymerase PhaZ (de Eugenio et al., 2007), and the proposed structural protein PhaD (Klinke et al., 2000). The promoter region regulating the PHA biosynthesis gene cluster is located upstream of the phaC1 gene (Timm and Steinbuchel, 1992) and is designated PphaC (Fig. 1B). Another gene clus- ter, comprising the genes encoding the PHA granule-associated proteins PhaF and PhaI, proposed to be involved in PHA biosyn- thesis regulation, is located downstream of the PHA biosynthesis gene cluster and transcribed in the opposite direction (Hoffmann and Rehm, 2004, 2005)(Fig. 1A). The promoter region regulat- ing the phaIF operon is designated PphaI. A model for PHA regulation was proposed for Pseudomonas oleovorans (Prieto et al., 1999), where a phaF knockout mutant was characterised. PhaF has then been proposed to be involved in the regulation of pha genes in P. oleovorans by repressing the expression of at least the phaC1 gene and the phaIF operon. According to this regulatory model, the phaF gene encodes a 0168-1656/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2007.11.007