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Proteomic Analysis of Herbaspirillum seropedicae Secreted Proteins
Chaves, D.F.S.; Monteiro, R.A.; Souza, E.M.; Yates, G.M.; Cruz, L.M.; Pedrosa, F.O
Departamento de Bioquímica e Biologia Molecular - Universidade Federal do Paraná - PR
The genome sequence of the nitrogen-fixing plant endophyte Herbaspirillum seropedicae has recently been completed by the GENOPAR Consortium. Analysis of this sequence revealed that this bacterium has type I, type II and type III secretion systems. Since secreted proteins play an important role in the development of the interaction between bacteria and eukaryotic cells this work is aimed at the identification of H. seropedicae secreted proteins.
To obtain secreted proteins, fresh cultures were centrifuged twice and the supernatants filtered through an 0.2-µm-pore-size filter to remove intact bacterial cells. The proteins were then precipitated using ammonium sulfate 70% (w/v) or TCA (12% final volume) and separated using various ranges of SDS-PAGE gels (12.5%; 8-25% and 20%). The proteins were visualized using colloidal comassie blue or silver stain. Proteins were excised from the gels, digested with trypsin and the peptide mass fingerprints were obtained using a Maldi-ToF/MS mass spectrometer. To identify the proteins, searches were performed using the monoisotopic mass profiles against the genome sequence of H. seropedicae.
Twenty-eight secreted proteins were identified in the supernatants of H. seropedicae cultures. Among them were proteins involved in the Sec-dependent secretion system, proteins involved in flagella assembly, outer membrane proteins belonging to the Omp family, ABC transport systems transmembrane proteins, proteins involved in the transport of amino acids and also some proteins involved in carbohydrate metabolism. Signal peptide proteins and peptidases were also identified. These results strongly suggest that type I and type II secretion systems are operational in H. seropedicae.
Supported by CNPq/PIBIC, CAPES and Fundação Araucária
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