Analysis of the Pseudomonas putida CA-3 proteome during growth on styrene under nitrogen-limiting and non-limiting conditions

Abstract

Pseudomonas putida CA-3 is a styrene-degrading bacterium capable of accumulating mediumchain-length polyhydroxyalkanoate (mclPHA) when exposed to limiting concentrations of a nitrogen source in the growth medium. Using shotgun proteomics we analysed global proteome expression in P. putida CA-3 supplied with styrene as the sole carbon and energy source under N-limiting (condition permissive for mclPHA synthesis) and non-limiting (condition non-permissive for mclPHA accumulation) growth conditions in order to provide insight into the molecular response of P. putida CA-3 to limitation of nitrogen when grown on styrene. A total of 1761 proteins were identified with high confidence and the detected proteins could be assigned to functional groups including styrene degradation, energy, nucleotide metabolism, protein synthesis, transport, stress response and motility. Proteins involved in the upper and lower styrene degradation pathway were expressed throughout the 48 h growth period under both nitrogen limitation and excess. Proteins involved in polyhydroxyalkanoate (PHA) biosynthesis, nitrogen assimilation and amino acid transport, and outer membrane proteins were upregulated under nitrogen limitation. PHA accumulation and biosynthesis were only expressed under nitrogen limitation. Nitrogen assimilation proteins were detected on average at twofold higher amounts under nitrogen limitation. Expression of the branched-chain amino acid ABC transporter was up to 16-fold higher under nitrogen-limiting conditions. Branched chain amino acid uptake by nitrogenlimited cultures was also higher than that by non-limited cultures. Outer membrane lipoproteins were expressed at twofold higher levels under nitrogen limitation. This was confirmed by Western blotting (immunochemical detection) of cells grown under nitrogen limitation. Our study provides the first global description of protein expression changes during growth of any organism on styrene and accumulating mclPHA (nitrogen-limited growth). INTRODUCTIONPseudomonas putida CA-3 has been reported to utilize styrene as a sole source of carbon and energy and to accumulate the biodegradable polymer medium-chainlength polyhydroxyalkanoate (mclPHA) when nitrogen (N) becomes limited in the growth medium (O'Connor et al., 1996;. These two abilities have been employed in the two-step chemo-biotechnological conversion of polystyrene, a non-biodegradable polymer, to mclPHA at laboratory scale (Ward et al., 2006). The process has been improved through the controlled feeding of N to the growth medium, which results in a twofold increase in biomass and a 1.4-fold increase in mclPHA accumulation (Goff et al., 2007).Molecular investigations of the styrene degradation pathway in this organism have been reported (Mooney et al., 2006b;O'Connor et al., 2001). Styrene metabolism in P. putida CA-3 proceeds via initial side chain oxidation and involves an upper pathway converting styrene to phenylacetic acid (PA) (O'Connor et al., 1995), and a lower pathway initiated via activation of PA to phenylace...

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