BIOCttlMIE, 1985, 67, 84%851 Linker mutagenesis in the gene encoding the periplasmic maltose-binding protein of E. coli. P. DUPLAY, H. BEDOUELLE, S. SZMELCMAN and M. HOFNUNG. Unitd de Programmation Moldculaire et Toxicologie Gdndtique, CNRS UA 271, INSERM U 163, Institut Pasteur, 28 rue du Dr. Roux, 75015 Paris. (Re~u le 4-2-1985, acceptd le 21-3-1985). R~sum6 -- Une sdrie de mutations, dans un plasmide multicopie portant le g~ne malE, ont dtd construites in vitro par insertion au hasard d'un octanucldotide de synthOse (adaptateur ou a linker ~) codant pour le site de reconnaissance de l'endonucldase BamHL Environ 25 % des plasmides testds avaient un site BamHI dans le gkne malE. La plupart des insertions obtenues dtaient accompagndes d'une petite ddldtion d'une longueur moyenne de 30 paires de base. Parmi les 21 mutants synthdtisant une protdine Male stable, 8 dtaient toujours capables de pousser sur milieu minimum maltose, line analyse prdliminaire de ces mutants r~vdle que certahles rdgions de la protdine ne doivent pas 6tre essentielles pour le transport du maltose. Mots-cl~s : MBP / mutag~n~se / adaptateur. Summary -- A plasmid carrying the malE gene, coding for the periplasmic maltose-binding protein of E. coli, was submitted to random mutagenesis by the insertion of a BamHI linker. About 25 % of the clones recovered had acquired a BamHI site in the gene malE. Most of the linker insertions were accompanied by small deletions with an average size of 30 base pairs. Among 21 mutants synthesizing a stable maltose binding protein, 8 were still able to grow on maltose. A preliminary analysis of these mutants indicates that certain regions of the protein may not be essential for maltose transport. Key-words : MBP / mutagenesis / linker. Introduction The maltose-binding protein (MBP) of E. coli is a multifunctional periplasmic protein involved in the specific, energy-dependent transport me- chanism of maltose and maltodextrins across the cell envelope; it also plays a key role as a receptor protein in chemotaxis towards maltose. This protein is encoded by the male gene, a member of the positively regulated malEFG operon [1 and ref. therein]. MBP exhibits specific binding of maltose and maltodextrins (I~ = 1 l.tM) and inte- racts with several proteins, including the LamB protein, the Tar protein and presumably one or more of the products encoded by the malK, malF and malG genes. MBP has been cristallized and the resolution of its three dimensional structure is in progress [2]. In order to study the relations- hips between structure and function, we have undertaken to mutate the malE gene, by random insertion of a BamHl linker, and to study the phenotypical and biochemical modifications of the resulting mutants. We present here a prelimi- nary report on this work.