The Major Phase-Variable Outer Membrane Protein of Escherichia coli Structurally Resembles the Immunoglobulin A1 Protease Class of Exported Protein and Is Regulated by a Novel Mechanism Involving Dam and OxyR

Abstract

Here we report the characterization of an Escherichia coli gene (agn43) which encodes the principal phase-variable outer membrane protein termed antigen 43 (Ag43). Theagn43 gene encodes a precursor protein of 107 kDa containing a 52-amino-acid signal sequence. Posttranslational processing generates an α43 subunit (predictedM r of 49,789) and a C-terminal domain (β43) with features typical of a bacterial integral outer membrane protein (predicted M r of 51,642). Secondary structure analysis predicts that β43 exists as an 18-stranded β barrel and that Ag43 shows structural organization closely resembling that of immunoglobulin A1 protease type of exoprotein produced by pathogenic Neisseria andHaemophilus spp. The correct processing of the polyprotein to α43 and β43 in OmpT, OmpP, and DegP protease-deficient E. coli strains points to an autocatalytic cleavage mechanism, a hypothesis supported by the occurrence of an aspartyl protease active site within α43. Ag43, a species-specific antigen, possesses two RGD motifs of the type implicated in binding to human integrins. The mechanism of reversible phase variation was studied by immunochemical analysis of a panel of well-defined regulatory mutants and by analysis of DNA sequences upstream of agn43. Evidence strongly suggests that phase variation is regulated by both deoxyadenosine methylase (Dam) and by OxyR. Thus, oxyR mutants are locked on for Ag43 expression, whereas dam mutants are locked off for Ag43 expression. We propose a novel mechanism for the regulation of phase switching in which OxyR competes with Dam for unmethylated GATC sites in the regulatory region of the agn43 gene.

Antigen 43 facilitates formation of multispecies biofilms

“…The difference can arguably be accounted for by the ability of the parent strain to express type 1 fimbriae. It should also be noted that in order to avoid phase variable expression of Ag43 the wild-type promoter of the flu gene (Hasman et al, 1999;Henderson and Owen, 1999) was replaced by the constitutive tet promoter in plasmid pKKJ128.…”

“…In separate studies, an outer membrane protein, Ag43, was characterized (Owen et al, 1987;Henderson et al, 1997). Recently, Ag43 was unambiguously identified as the product of the flu gene (Hasman et al, 1999;Henderson and Owen, 1999). It was further established that autoaggregation of cells takes place through an Ag43±Ag43 interaction (Hasman et al, 1999).…”

“…Also, expression is phase variable, with cells shifting from Ag43 1 to Ag43 2 and vice versa at about 10 23 per cell per generation. The phase variation results from the combined regulation of the flu gene by the Dam methylase and OxyR (Warne et al, 1990;Henderson et al, 1997;Henderson and Owen, 1999;Haagmans and van der Woude, 2000). Ag43 is a member of the family of autotransporter proteins (Henderson et al, 1998).…”

Phase variation controls expression of Salmonella lipopolysaccharide modification genes by a DNA methylation‐dependent mechanism

“…In the agn43 regulatory region there is only one binding site for OxyR that acts as a repressor in this system. Methylation of the GATC sequences abrogates binding, allowing agn43 expression (Henderson and Owen, 1999;Haagmans and van der Woude, 2000). Dam and OxyR also control expression of the phage Mu mom gene, but whether expression phase varies is not known (Hattman and Sun, 1997).…”

Phase variable restriction–modification systems in Moraxella catarrhalis

“…Regulation of virulence determinant expression by methylation has been well documented. Dam methylation a¡ects Pap pili [44] and Ag43 [28] expression in E. coli, virulence gene expression in Salmonella typhimurium [45] and the rate of phase variation of capsular polysaccharide expression in N. meningitidis [46] (this is not seen in all phase variable systems in N. meningitidis and is not the result of mismatch repair de¢ciencies [47]). It has also been suggested that phase variation of R^M activity is associated with antigenic variation in M. pulmonis [25,26].…”