Comparison of proteins AntA (94 aa) and AntB (209 aa) with each other revealed 62% amino acid identities near their N termini. and most strains of the phylogenetic group B2. Sequencing of a 1.1-kb portion ofibrABrevealed that the othereib-positive strains diverge by 0.1% from ECOR-9, whereaseib-negative ECOR-47 diverges by 16%. TheEscherichia coliimmunoglobulin (Ig)-binding (Eib) proteins are members of a larger family of surface-exposed bacterial proteins that includes YadA ofYersinia(39,44,45), UspAII ofMoraxella(3,4), and DsrA ofHaemophilus ducreyi(15). The Eib proteins have several phenotypic features in common with these proteins, such as the ability to impart resistance to human serum complement and a UNC0379 UNC0379 tendency to exist as highly stable multimers. In addition to the properties shared with other members of this protein family, the Eib proteins can bind Igs such as IgA and/or the Fc fragment of human IgG (IgG Fc) in a nonimmune manner (40,41). Theeibgenes are strongly expressed as the cells enter stationary phase at 37C (42). The Eib proteins were originally identified in six of the 72 strains of theE. colireference (ECOR) strain Rabbit Polyclonal to F2RL2 collection (33). One of these six strains, ECOR-9, was found to produce several distinct Ig-binding proteins, each encoded by a different member of a set of related prophages. Four genes,eibA,eibC,eibD, andeibE, were cloned from ECOR-9. In multicopy each imparted Ig-binding activity and enhanced serum resistance to a test recipient. In addition a series of lysogenic derivatives ofE. coliC was established usingeib-bearing phages derived from ECOR-9 (P-EibB, P-EibC, P-EibD, and P-EibE) (40). The derived lysogens, bearing from one to three of the prophages, expressed little or no Eib, in sharp contrast to the parental ECOR-9 lysogen, suggesting that ECOR-9 has an expression-enhancing activity that the derived lysogens lack. The goal UNC0379 of the present study was to clone from ECOR-9 a gene or genes that would activate expression of Ig-binding activity in the lysogens. == MATERIALS AND METHODS == == Strains and culture conditions. == TheE. coliK-12 strain DH5 was used for cloning of all constructs. All lysogens were derivatives ofE. colistrain C, a nonrestricting strain (6), and are listed (see Table2). Cultures used for protein extraction were grown in UNC0379 Luria-Bertani (LB) broth for 18 to 24 h at 37 with agitation and harvested by centrifugation at 4C. LB broth containing ampicillin, 50 g per ml, was used for UNC0379 maintenance of pUC21 derivatives. LB broth containing kanamycin, 50 g per ml, was used to maintain pOK12 derivatives. == TABLE 2. == E. coliC derivative strains See reference40. See Materials and Methods. == Protein extraction and Ig binding. == Preparation of cell extracts, determination of protein concentration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotting were as described previously (42). Our standard immunoblotting procedure entails a one-step incubation with nonimmune antibody, such as the Fc fragment of human IgG (IgG Fc) conjugated with horseradish peroxidase (HRP). It is important to note that there is no incubation with primary antibody specifically directed against an antigen. Purified Fc fragment of human IgG conjugated with HRP (IgG Fc-HRP) (Rockland) was used at a concentration of 50 ng of antibody per ml. == DNA cloning and analysis. == Techniques used for DNA isolation, cloning, and sequence analysis involve minor modifications of those indicated elsewhere.