Comprehensive Proteomics Analysis of Polyhydroxyalkanoate (PHA) Biology in Pseudomonas putida KT2440: The Outer Membrane Lipoprotein OprL is a Newly Identified Phasin

Pseudomonas putida KT2440 is an important bioplastic-producing industrial microorganism capable of synthesizing the polymeric carbon-rich storage material, polyhydroxyalkanoate (PHA). PHA is sequestered in discrete PHA granules, or carbonosomes, and accumulates under conditions of stress, for example, low levels of available nitrogen. The pha locus responsible for PHA metabolism encodes both anabolic and catabolic enzymes, a transcription factor, and carbonosome-localized proteins termed phasins. The functions of phasins are incompletely understood but genetic disruption of their function causes PHA-related phenotypes. To improve our understanding of these proteins, we investigated the PHA pathways of P.putida KT2440 using three types of experiments. First, we profiled cells grown in nitrogen-limited and nitrogen-excess media using global expression proteomics, identifying sets of proteins found to coordinately increase or decrease within clustered pathways. Next, we analyzed the protein composition of isolated carbonosomes, identifying two new putative components. We carried out physical interaction screens focused on PHA-related proteins, generating a protein-protein network comprising 434 connected proteins. Finally, we confirmed that the outer membrane protein OprL (the Pal component of the Pal-Tol system) localizes to the carbonosome and shows a PHA-related phenotype and therefore is a novel phasin. The combined datasets represent a valuable overview of the protein components of the PHA system in P.putida highlighting the complex nature of regulatory interactions responsive to nutrient stress. [Display omitted] •Comprehensive analysis of Pseudomonas putida KT2440 an important industrial microorganism.•Expression proteomics identified proteins upregulated under PHA-producing conditions. Analysis of the PHA granule mapped a newly identified phasin.•Protein interaction screen found links between the phasins PhaI and PhaF, and the pyruvate dehydrogenase E1 subunit. Pseudomonas putida KT2440 is capable of producing the biodegradable bioplastic polyhydroxyalkanoate (PHA). To better understand the pathways responsible for PHA metabolism, several types of proteomics experiment were carried out. The proteome response to low nitrogen was examined, followed by cataloguing proteins that physically associate with the PHA storage granule, and a protein interaction network for the key PHA enzyme. The work confirmed the importance of a newly identified phasin (OprL)