Olaf Mol

Membrane association of FtsY, the E. coli SRP receptor

FtsY, the Escherichia coli homologue of the eukaryotic SRP receptor (SRα), is located both in the cytoplasm and in the inner membrane of E. coli. Similar to SRα, FtsY consists of two major domains: a strongly acidic N‐terminal domain (A) and a C‐terminal GTP binding domain (NG) of which the crystal structure has recently been determined. The domains were expressed both in vivo and in vitro to examine their subcellular localization. The results suggest that both domains associate with the membrane but that the nature of the association differs.

Escherichia coli periplasmic chaperone FaeE is a homodimer and the chaperone-K88 subunit complex is a heterotrimer

The interaction of FaeE, a periplasmic chaperone involved in K8B biosynthesis, and the major fimbrial subunit FaeG was Investigated. The genes encoding the two proteins were subcloned together in the expression vector pINIIIA1, Cells expressing the sub‐cloned genes accumulated in their periplasm a complex of FaeE and FaeG. This complex was purified by isoelectric focusing and anion‐exchange fast‐protein liquid chromatography. SOS‐PAGE, native gel etectrophoresis, immunoblotting and determination of the N‐terminal amino acid sequences and the molar ratio of the W‐terminal amino acid residues revealed that the complex is a heterotrimer consisting of two molecules of FaeE and one molecule of FaeG. The periplasmic chaperone FaeE was purified from the periplasm of cells expressing only the subcloned faeE gene. Gel filtration, protein cross‐linking analysis and a biophysical approach in which the rotation diffusion coefficient of the purified FaeE was determined led to the conclusion that the native FaeE chaperone is a homodimer.

The Escherichia coli K88 periplasmic chaperone FaeE forms a heterotrimeric complex with the minor fimbrial component FaeH and with the minor fimbrial component Fael

K88ab fimbriae are long polymeric protein structures mainly composed of FaeG proteins. The Escherichia coli K88 periplasmic chaperone FaeE is a homodimer and forms a heterotrimeric complex with the K88 major fimbrial component FaeG in the periplasm. In this study the direct interaction of FaeE and the minor K88 fimbrial subunits FaeH and FaeI were investigated. The faeH gene and the faeI gene were subcloned in a pINIIIA1-derivative vector containing the faeE gene. SDS-PAGE using normal and gradient gels and immunoblotting revealed that the subcloned genes were expressed in the periplasm. Analyses of periplasmic fractions by native gel electrophoresis and isoelectric focusing (IEF) showed that FaeE and FaeH, as well as FaeE and FaeI formed protein complexes. These complexes were isolated and purified by FPLC or IEF and native gel electrophoresis. The stoichiometry of the proteins in these complexes was studied by automated Edman degradation and gel image analysis. The results showed that FaeE and FaeH, and FaeE and FaeI formed heterotrimeric E2H and E2I complexes, respectively. In addition to the E2H complex, cells expressing FaeE and FaeH accumulated unbound FaeH in their periplasm. In contrast to the E2G complex, the purified E2H complex was not stable and was partly dissociated in the experimental conditions used, suggesting that the interaction between FaeE and FaeH is not as strong as the interaction of FaeE and FaeG.

The N‐terminal ‐barrel domain of the Escherichia coli K88 periplasmic chaperone FaeE determines fimbrial subunit recognition and dimerization

The K88 periplasmic chaperone FaeE is a homodimer, whereas the K99 chaperone FanE is a monomer. The structural requirements for dimerization of the K88 fimbrial periplasmic chaperone and for fimbrial subunit‐binding specificity were investigated by analysis of mutant chaperones. FaeE contains a C‐terminal extension of 19 amino acid residues when compared to FanE and most other fimbrial chaperones. A C‐terminal truncate of the K88 chaperone FaeE was constructed that lacked 19 C‐terminal amino acid residues. Expression and complementation experiments revealed that this C‐terminal shortened chaperone was still functional in binding the K88 major subunit FaeG and K88 biosynthesis. Two hybrid chaperones were constructed. Each hybrid protein contained one ‐barrel domain of FaeE and the other ‐barrel domain of FanE (Fae/FanE or Fan/FaeE, respectively). Expression and complementation experiments revealed that the Fae/FanE but not the Fan/FaeE hybrid chaperone was functional in the formation of K88 fimbriae. The Fan/FaeE hybrid chaperone was active in the biosynthesis of K99 fimbriae. The truncated FaeE mutant chaperone and the hybrid Fae/FanE chaperone were able to form stable periplasmic protein complexes with the K88 major fimbrial subunit FaeG. Cross‐linking experiments suggested that the C‐terminal shortened chaperone and the Fae/FanE hybrid chaperone were homodimers, as is the wild‐type K88 chaperone. Altogether, the data suggested that the N‐terminal ‐barrel domain of a fimbrial chaperone determines subunit specificity. In the case of the K88 periplasmic chaperone, this N‐terminal domain also determines dimerization of the protein.

Functioning of the pCLODF13 Encoded BRP

Bacteriocin release proteins (BRP’s) or “lysis” proteins are small membrane associated proteins required for the translocation of bacteriocins across the cell envelope of E. col. (De Graaf & Oudega, 1986). The gene encoding the “lysis” protein is transcribed from the same promoter as the gene encoding its corresponding bacteriocin (De Graaf & Oudega, 1986). Subcloning of several “lysis” protein genes in expression vectors under separate promoter control has facilitated the study of their structure and function (Pugsley & Schwartz, 1983; Altieri et a.., 1986; Luirink et a.., 1987a; Cavard et a.., 1989).