Sevec Szmelcman

Silent and functional changes in the periplasmic maltose-binding protein of Escherichia coli K12: I. Transport of maltose

The malE gene encodes the periplasmic maltose-binding protein (MBP). Nineteen mutations that still permit synthesis of stable MBP were generated by random insertion of a BamHI octanucleotide into malE and six additional mutations by in-vitro recombinations between mutant genes. The sequence changes were determined; in most cases the linker insertion is accompanied by a small deletion (30 base-pairs on average). The mutant MBP were studied for export, growth on maltose and maltodextrins, maltose transport and binding, and maltose-induced fluorescence changes. Sixteen mutant MBP (out of 21 studied in detail) were found in the periplasmic space: 12 of them retained a high affinity for maltose, and 10 activity for growth on maltose. The results show that several regions of MBP are dispensable for stability, substrate binding and export. Three regions (residues 207 to 220, 297 to 303 and 364 to 370) may be involved in interactions with the MalF or MalG proteins. A region near the C-terminal end is important for maltose binding. Two regions of the mature protein (residues 18 to 42 and 280 to 296) are required for export to, or solubility in, the periplasm.

Genetic approach to the role of tryptophan residues in the activities and fluorescence of a bacterial periplasmic maltose-binding protein

The periplasmic maltose-binding protein (MBP or MalE protein) of Escherichia coli is an essential element in the transport of maltose and maltodextrins and in the chemotaxis towards these sugars. On the basis of previous results suggesting their possible role in the activity and fluorescence of MBP, we have changed independently to alanine each of the eight tryptophan residues as well as asparagine 294, which is conserved among four periplasmic sugar-binding proteins. Five of the tryptophan mutations affected activity. In four cases (substitution of Trp62, Trp230, Trp232 and Trp340), there was a decrease in MBP affinity towards maltose correlated with modifications in transport and chemotaxis. According to the present state of the 2.3 A three-dimensional structure of MBP, all four residues are in the binding site. Residues Trp62 and Trp340 are in the immediate vicinity of the bound substrate and appear to have direct contacts with maltose; this is in agreement with the drastic increases in Kd values (respectively 67 and 300-fold) upon their substitution by alanine residues. The modest increase in Kd (12-fold) observed upon mutation of Trp230 would be compatible with the lesser degree of interaction this residue has with the bound substrate and the idea that it plays an indirect role, presumably by keeping other residues involved directly in binding in their proper orientation. Substitution of Trp232 resulted in a small increase in Kd value (2-fold) in spite of the fact that this residue is the closest to the ligand of the tryptophan residues according to the three-dimensional model. In the fifth case, replacement of Trp158, which is distant from the binding site, strongly reduced the chemotactic response towards maltose without affecting the transport parameters or the sugar-binding activities of the mutant protein. Trp158 may therefore be specifically implicated in the interaction of MBP with the chemotransducer Tar, but this effect is likely to be indirect, since Trp158 is buried in the structure of MBP. Of course, some structural rearrangements could be responsible in part for the effects of these mutations. The remaining four mutations were silent. The corresponding residues (Trp10, Trp94, Trp129 and Asn294) are all distant from the sugar-binding site on the crystallographic model of MBP, which is in agreement with their lack of effect on binding. In addition, our results show that they play no role in the interactions with the other proteins of the maltose transport (MalF, MalG or MalK) or chemotaxis (Tar) systems.(ABSTRACT TRUNCATED AT 400 WORDS)

Silent and functional changes in the periplasmic maltose-binding protein of Escherichia coli K12: II. Chemotaxis towards maltose☆

We examined the chemotactic behavior of ten Escherichia coli mutants able to synthesize a modified periplasmic maltose-binding protein (MBP) retaining high affinity for maltose. Eight were able to grow on maltose (Mal+), two were not (Mal-). In the capillary assay six out of eight of the Mal+ strains showed an optimal response at the same concentration of maltose as the wild-type strain; the amplitude of the response was strongly reduced in two Mal+ mutants and partially affected in one. The amplitude of the chemotactic response of the two Mal- strains was at least equal to that of the wild type, so that the chemotactic and transport functions of MBP were dissociated in these two cases. We define two regions of the protein (residues 297 to 303 and 364 to 369), that are important both for the chemotactic response and for transport, and one region (residues 207 to 220) that is essential for transport but dispensable for chemotaxis. Interestingly, some regions that were found to be inessential for transport are also dispensable for chemotaxis.

Palindromic units from E. coli as binding sites for a chromoid-associated protein

Several hundred copies of a highly conserved extragenic palindromic sequence, 20–40 nucleotides long, exist along the chromosome of E. coli and S. typhimurium. These have been defined as palindromic units (PU) or repetitive extragenic palindromes (REP). No general function for PUs has been identified. In the present work, we provide data showing that a protein associated with a chromoid extract of E. coli protects PU DNA against exonuclease III digestion. This provides the first experimental evidence that PU constitutes binding sites for a chromoid‐associated protein. This result supports the hypothesis that PUs could play a role in the structure of the bacterial chromoid.

Linker mutagenesis in the gene encoding the periplasmic maltose-binding protein of E. coli.

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.