The Escherichia coli AIDA-I autotransporter undergoes cytoplasmic glycosylation independently of export.

The Escherichia coli adhesin involved in diffuse adherence (AIDA-I) is an outer membrane autotransporter protein and one of the few glycosylated proteins found in Escherichia coli. O-glycosylation is mediated by the product of the aah gene, which codes for a heptosyltransferase that uses ADP-glycero-manno-heptose precursors from the LPS biosynthesis pathway. Little else is known about Aah and mechanisms involved in modification of AIDA-I. We observed that Aah is mainly found in an insoluble fraction and, by deletion of the AIDA-I signal sequence or by blocking sec-translocation machinery with sodium azide, we showed that glycosylation occurs in the cytoplasm of bacteria independently of secretion. Since AIDA-I harbors an N-terminal extension in its signal sequence, we wondered whether glycosylation requires this unusual sequence. We observed that, while deletion of the N-terminal extension affected the expression level of AIDA-I, the protein was still exported to the outer membrane and glycosylated. Modification of a secreted protein in the cytoplasm raises several mechanistic questions.