ANALYSIS OF POLYHYDROXYALKANOATE GRANULE ASSOCIATED PROTEINS, PHASINS, IN Pseudomonas extremaustralis

CATONE M.V; MEZZINA MP; LÓPEZ, NANCY I

Rosario, Santa Fe

Congreso; IX Congreso de la Sociedad Argentina de Microbiología General (SAMIGE); 2013

SAMIGE

Pseudomonas extremaustralis produces mainly polyhydroxybutyrate (PHB), a short chain length polyhydroxyalkanoate (sclPHA), infrequently found in Pseudomonas, and small amounts of medium chain length polyhydroxyalkanoates (mclPHA). Previous works allowed to identify different gene clusters involved in PHA production. Two gene clusters are related to PHB biosynthesis, phbRBAC containing the class I PHA synthase (phbC) and phbFPX, that comprises two genes encoding phasin like proteins (phbP and phbF). The mclPHA cluster, phaC1ZC2D, includes two classII PHA synthases, a depolymerase, and a regulator. The phaF and phaI, encoding mclPHA phasins, in P. extremaustralis are not located downstream phaD because of the insertion of seven ORF not related to PHA synthesis. Phasins are amphipathic PHA granule-associated proteins (GAPs), involved in stabilization of carbon storage granules. In this work, four different GAPs involved in scl and mclPHA production in P. extremaustralis genome were analyzed. Bioinformatic analysis were performed using BLAST, ClustalW and SEED-Viewer included in the RAST server. Phylogenetic three construction was conducted using MEGA 4. Experimental analysis included Western blot assays and the identification of phasins by high resolution mass spectrometry in PHA granule isolations. The mclPHA phasins showed high identity with other similar proteins found in Pseudomonas species. By contrast, PhbP and PhbF, related to PHB metabolism, presented high identity with Azotobacter strains and bacteria belonging to b-Proteobacteria. Phylogenetic analysis allowed to infer that these genes have different origins. PhaF showed the presence of 16 histone like domain AAKP. This domain showed a variable number of repetitions among different Pseudomonas species, finding 12 repetitions in the model bacterium P. putida KT2440. PhaF and PhaI shared 31% of identity while the two phasins involved in PHB metabolism shared only 14% of identity. Sequence comparisons of phasins involved in different kind of PHA production showed less identity. Western blot analysis performed with an antibody against PhaP from Azotobacter sp. FA8, a PHB producer, allowed the detection of PhbP of P. extremaustralis, consistently with the high identity between these proteins. SDS-PAGE of PHA granules extracted from stationary phase cultures from the wild type and a mutant strain, unable to produce mclPHA, showed the presence of PhaI, involved in the synthesis of mclPHA and PhbP, which represented the major band in the gel. PhbF and PhaF, were not detected in the granules. Results indicated that PHA granules are mainly coated by PhbP and the mclPHA phasin, PhaI, was also granule associated even in a mutant strain for mclPHA production. Studies of phasins involved in controlling number and size of granules could help to design strategies leading to improve polymer production in P. extremaustralis, a suitable microorganism for the synthesis of different kind of PHA.