CONSTRUCTION AND 1ST CHARACTERIZATION OF 2 RECIPROCAL HYBRIDS BETWEENLAMB FROM ESCHERICHIA-COLI K12 AND KLEBSIELLA-PNEUMONIAE

The LamB proteins from Klebsiella pneumoniae and Escherichia coli K12 were previously shown to be highly homologous. The most conserved part s correspond to the N-proximal third and to the transmembranous portio ns of the molecule, while the variability occurred essentially within regions exposed to the cell surface or to the periplasm. Since the two proteins displayed identical in vitro trimer stability and in vivo po re properties, we tested whether the N-terminal parts of the two prote ins could be exchanged and still allow the formation of stable and fun ctional maltoporins. For that purpose, we expressed the LamB protein f rom K. pneumoniae in E. coli K12, and constructed two reciprocal hybri ds between LamB from E. coli K12 and LamB from K. pneumoniae. The firs t hybrid (LamBE.c.-K.p.) is composed of residues 1 to 183 from LamBE.c . followed by residues 184 to 404 from LamBK.p. The second one compris es residues 1 to 183 from LamBK.p., followed by residues 184 to 421 fr om LamBE.c. (LamBK.p.-E.c.). Both hybrid proteins were correctly incor porated in the outer membrane of E. coli K12. Like the two parental La mB proteins, the two hybrids could be purified by affinity chromatogra phy on a starch-sepharose column. The LamBE.c.-K.p. hybrid formed high ly stable trimers, but was strongly impaired in its in vivo maltose tr ansport function (15 % of the wild-type level). The trimers formed by LamBK.p.-E.c. hybrid were less stable, but could be detected on the su rface of intact cells by four anti-LamBE.c. monoclonal antibodies. Thi s hybrid was also affected in its in vivo maltose transport function ( 30 % of the wild-type level). As expected from the location of the res idues critical for phage adsorption, both proteins had lost the phage receptor activity of the E. coli K12 LamB protein. We also examined wh ether LamBE.c. could form heterotrimers with LamBK.p., LamBK.p.-E.c., and LamBE.c.-K.p. In no case were heterotrimers detected, indicating t hat both terminal parts of the LamB protein are involved in homotrimer formation. All these data suggest that trimer formation and activity involve rare variable residues in the conserved regio s and/or variabl e regions.