The gene cluster of subsp. (Ecc) encodes the proteins of the type II or general secretory pathway (GSP) apparatus which is required for secretion of pectinase and cellulase. In this study, fusions between Ecc genes and the topology probe were constructed. The ability of Out protein domains to export BlaM across the cytoplasmic membrane in both and the cognate host was utilized to confirm the computer-predicted cytoplasmic membrane topology of OutC and OutF. When was fused to , the resulting phenotype suggested that the majority of OutC is targeted to the periplasm, typical of a type II bitopic conformation in the cytoplasmic membrane. In contrast, for the gene product, three transmembrane regions were identified which connect a large N-terminal cytoplasmic domain, a smaller periplasmic domain, and a large cytoplasmic loop. Fusions between and and were used to further substantiate the locations of these gene products in the outer membrane and the cytoplasm respectively. The data derived suggest that a number of the Out apparatus components possess domains in the cytoplasm and/or the periplasm with potential for protein-protein interactions which facilitate the secretion of periplasmic enzyme intermediates across the outer membrane to the external milieu.


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