Implication of Omp85 on the secretion and insertion of the S-layer protein from Thermus thermophilus

FEDERICO ACOSTA; JOSE BERENGUER

Congreso; IX Meeting of the Spanish Scientific Network on Extremophilic Microorganisms; 2009

Implication of Omp85 on the secretion and insertion of the S-layer protein from Thermus thermophilus Federico Acosta y José Berenguer Centro de Biología Molecular Severo Ochoa. Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas. β-Barrel outer membrane proteins are present in Gram-negative bacteria, mitochondria and chloroplasts. It has been described the implication of proteins belonging to the Omp85, a beta-barrel protein itself, in the insertion and assembly of such β-Barrel outer membrane protein (OMP) through a poorly known mechanism. In the system so far analyzed, a C-terminal signature sequence found in many OMP beta-barrel proteins binds a N-terminal periplasmic extension of Omp85 which includes a domain called POTRA (polypeptide transport associated). Although this interaction could be species-specific, the C-terminal signature of the secreted OMP is usually well conserved, including a C-terminal Phe residue. The cell envelope of the thermophile Thermus thermophilus is mulilayered, comprising a cytoplasmatic membrane, a thin cell wall layer, and an outer membrane/S-layer envelope bound to the secondary cell wall polymers. The S-layer is formed by subunits of SlpA (Surface layer protein A), a protein which has mixed characters common to both S-layers and outer membrane proteins. Although SlpA has a sec dependent signal peptide for its secretion, it is not known the mechanism underlying its secretion and precise integration in the OM envelope. Analysis of SlpA proteins sequences from different strains of T. thermophilus revealed a C-terminal signature sequence, similar to the consensus sequence used for secretion of OM proteins in Gram negative bacteria. On the other hand, a homologue to Omp85 has been described in this organism. Therefore, we hypothesized that SlpA could require this Omp85 homologue for its incorporation to the outer membrane. To analyze this hypothesis we have generated slpA and omp85 mutants that produce proteins with changes or deletions at their respective C-terminal sequence. Our results show that proper location of SlpA depends on its C-terminal sequence, and that Omp85 is implicated in this process.