PROTEOMICS OF POLYHYDROXYBUTYRATE GRANULES REVEALS THE PRESENCE OF DIFFERENT KIND OF SYNTHASES IN Pseudomonas extremaustralis.

CATONE, M.V.; RUIZ, J.A.; CASTELLANOS, M.; SEGURA, D.; ESPIN, G.; LÓPEZ, N.I.

Mar del Plata

Congreso; VIII Congreso Argentino de Microbiología General Samige del Bicentenario; 2012

PROTEOMICS OF POLYHYDROXYBUTYRATE GRANULES REVEALS THE PRESENCE OF DIFFERENT KIND OF SYNTHASES IN Pseudomonas extremaustralis Catone*1 M.V.; Ruiz1 J.A.; Castellanos2 M.; Segura2 D.G.; Espin E.G.2 and López1 N. I. 1Dpto. de Qca Biológica, FCEyN, UBA 2IBT, UNAM, México. (*mcatone@qb.fcen.uba.ar) Polyhydroxyalkanoates (PHAs) are prokaryotic storage compounds that are accumulated intracellularly in form of granules. PHAs can consist of short-chain-length hydroxyalkanoic acids (PHASCL) such as poly(3-hydroxybutyrate) (PHB) or medium-chain-length monomers (PHAMCL) as poly(3-hydroxyoctanoate) which are synthesized by different classes of PHA synthases, the main enzymes involved in the polymer production. Pseudomonas extremaustralis DSM17835 is an Antarctic species able to produce PHAs in the form of PHB. However, the genome of this species contains several groups of genes involved in PHA metabolism including those related with PHAmcl production. In this work, we analyzed the transcription of all PHA synthases and the granule-bound proteins in P. extremaustralis. The phaRBAC cluster involved in PHB biosynthesis includes a class I phaC synthase or polymerase. In addition, we also identified a PHAmcl cluster phaC1ZC2D that differs of the typical PHAmcl cluster of Pseudomonas species due to the insertion of seven genes not related to PHA synthesis, between phaD and phaFI. This cluster contains two different class II polymerases, encoded by phaC1 and phaC2, whose functionality has been previously demonstrated by complementation assays. The genome of P. extremaustralis also presented other group of PHA genes that include two phasins like proteins, amphiphilic proteins located on surfaces of the granules, and a putative PHB depolymerase. The PHB granules were isolated from the crude extract of P. extremaustralis by using a glycerol gradient. Proteins were separated by SDS-PAGE and identified by high resolution mass spectrometry. As expected, the analysis of the protein sequences demonstrated the presence of class I PhaC synthase, responsible of PHB biosynthesis along with a putative PHB depolymerase and a phasin like PhbP protein. In addition, we found a class II PhaC synthase and the phasin like PhaI, both proteins associated to PHAmcl synthesis. Real time RT-PCR experiments were performed in order to study the expression of the three phaC synthases present in this strain employing RNA extracted from cultures grown with sodium octanoate or glucose as carbon source. The results shown that the different phaC genes are expressed at similar levels, in agreement with the proteomic assay of PHB granules. These results suggest that the absence of PHAmcl production is not due to differences in the transcription or expression of class II phaC synthases. We hypothesize that other factors could explain the inability of P. extremaustralis to accumulate PHAmcl, such as kinetic factors like substrate competition among different synthases or monomer availability.