Genetic analysis of sequences in maltoporin that contribute to binding domains and pore structure.
Maltoporin (LamB protein) is a maltodextrin transport protein in the outer membrane of Escherichia coli with binding sites for bacteriophage lambda and maltosaccharides. Binding of starch by bacteria was found to inhibit swarming of Escherichia coli in soft agar plates; the inhibition was dependent on the maltodextrin affinity of maltoporin. On the basis of this observation, chemotactic cell-sorting techniques were developed for the isolation and analysis of mutants with an altered starch-binding phenotype. Fifteen lamB mutations generated by hydroxylamine and linker mutagenesis, as well as spontaneous mutations, were analyzed. The effects of the mutations on starch and lambda-binding, as well as transport specificity, were assayed. Mutations that affect residues near 8 to 18, 74 to 82, and 118 to 121 were found to affect starch binding and maltodextrin-selective functions strongly, confirming and extending previous results with substitutions at these regions. Substitutions and insertions in two previously undefined regions in the protein, in or near residues 194 and 360, also resulted in defects in maltodextrin-specific functions and indicate that C-terminal parts of the protein also contribute to the discontinuous binding and pore domains. There was a detectable transport defect in all binding-affected mutants, and one mutation caused near-total pore blocking towards both maltose and nonmaltoside. The highly discontinuous phage lambda-binding site was affected by mutations near residues 9 and 10 and 194, as well as previously established regions near residues 18, 148 to 165, 245 to 259, and 380 to 400. The significance of these mutations is discussed in the context of a model of the functional topology of maltoporin. The additional role of regions near residues 10 and 120 in maltoporin assembly, as well as starch binding, was suggested by the temperature-sensitive biogenesis of maltoporin in strains with one- or two-codon insertion at these sites.
Heine
H G
HG
Department of Microbiology, University of Sydney, New South Wales, Australia.
Francis
G
G
Lee
K S
KS
Ferenci
T
T
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D010582:Bacteriophage lambda / Q000378:metabolism; D001692:Biological Transport; D002465:Cell Movement; D002633:Chemotaxis; D004262:DNA Restriction Enzymes; D003912:Dextrins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D005798:Genes, Bacterial; D007785:Lactose / Q000378:metabolism; D008320:Maltose / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D018272:Porins; D011487:Protein Conformation; D011991:Receptors, Virus / Q000235:genetics* / Q000378:metabolism; D013213:Starch / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D003912:Dextrins; D018272:Porins; D011991:Receptors, Virus; C016532:maltoporins; D008320:Maltose; D013213:Starch; D004262:DNA Restriction Enzymes; D007785:Lactose