Open AccessThe N‐terminal third of the BinB subunit from the Bacillus sphaericus binary toxin is sufficient for its interaction with midgut receptors in Culex quinquefasciatus
Author(s) -
Romão Tatiany Patrícia,
deMeloNeto Osvaldo Pompílio,
SilvaFilha Maria Heleeves Lobo
Publication year - 2011
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2011.02325.x
Subject(s) - bacillus sphaericus , culex quinquefasciatus , midgut , biology , toxin , microbiology and biotechnology , pore forming toxin , protein subunit , bacillales , genetics , bacteria , microbial toxins , ecology , bacillus subtilis , gene , larva , aedes aegypti
Heterodimeric binary (Bin) toxin, the major insecticidal protein from Bacillus sphaericus , acts on Culex quinquefasciatus larvae through specific binding to the midgut receptor Cqm1, a role mediated by its 448‐amino‐acid‐long BinB subunit. The molecular basis for receptor recognition is not well understood and this study attempted to identify protein segments and amino acid motifs within BinB that are required for this event. First, N‐ and C‐terminally truncated constructs were evaluated for their capacity to bind to native Cqm1 through pull‐down assays. These showed that residues N33 to L158 of the subunit are required for Cqm1 binding. Nine different full‐length mutants were then generated in which selected blocks of three amino acids were replaced by alanines. In new pull‐down assays, two mutants, in which residues 85 IRF 87 and 147 FQF 149 were targeted, failed to bind the receptor. Competition binding assays confirmed the requirements for the N‐terminal 158 residues, and the 147 FQF 149 epitope, for the mutant proteins to compete with native Bin toxin when binding to membrane fractions from the insect midgut. The data from this work rule out the involvement of C‐terminal segments in receptor binding, highlighting the need for multiple elements within the protein's N‐terminal third for it to occur.