A chimeric nucleotide‐binding protein, encoded by a hisP–malK hybrid gene, is functional in maltose transport in Salmonella typhimurium

Abstract

We have isolated a hybrid gene, composed of the first 455 nucleotides of hisP and nucleotides 275-1107 of malK, the genes coding for the nucleotide-binding components of the high-affinity transport systems for histidine and maltose in Salmonella typhimurium, respectively. The fusion had occurred by recombination within 11 homologous base pairs located between the two DNA fragments. In the chimeric protein peptidic motifs A and B, proposed to be part of the nucleotide-binding fold, originate from HisP and MalK, respectively. Plasmid pES42-39, harbouring the hybrid gene, was shown to complement only a malK mutation but failed to complement a hisP deletion mutation. The chimeric protein was identified by immunoblotting as a protein with an apparent molecular mass of 49kDa. Removal of the C-terminal 77 amino acid residues from the chimeric protein resulted in the loss of function in transport. In contrast, 51 amino acid residues could be removed from the C-terminus of wild-type MalK without any effect. Upon overproduction the chimeric protein, as wild-type MalK, inhibited expression of the malB regulon. However, both truncated proteins, when overproduced, did not exhibit this activity. Based on these results, a tentative model of the functional domains of MalK is presented.

MalFGK complex assembly and transport and regulatory characteristics of MalK insertion mutants

“…3. MalK derivatives that were compromised in transport or regulation generally followed a pattern suggested in other reports (5,9,20): most mutants with an insert in the N-terminal portion of the protein were severely defective in maltose transport; mutants defective in MalT and/or EIIA glc regulation had inserts in the C-terminal half of the protein. The exceptions to these generalizations provide new structural clues about MalK.…”

“…As most of these mutations map in the promoter-distal half of the gene, one might conclude from this that there are no essential interactions between the C-terminal portion of MalK and integral membrane proteins MalF and MalG. In contrast, deletion mutant studies by Schneider and Walter (20) suggest that residues 293 to 317 of MalK are critical for interaction with MalF and MalG. The fact that none of our malK-lacZ fusions (expressing C-terminally truncated MalK derivatives) was able to complement a malK deletion for transport also is consistent with some requirement of the C-terminal region of MalK for maltose transport.…”

“…This interaction downregulates the transport activity of the transport complex (5,9). Previously, regions of MalK involved in maltose transport and in regulation by MalT and EIIA glc have been examined by using a variety of point and deletion mutants (5,9,20). However, the three-dimensional structure of MalK is not known and its intra-and intermolecular interactions remain largely uncharacterized.…”

ATP Modulates Subunit-Subunit Interactions in an ATP-binding Cassette Transporter (MalFGK2) Determined by Site-directed Chemical Cross-linking

“…Plasmidencoded mal gene expression was induced by the addition of 0.5 or 1 mM isopropyl-␤-thiogalactoside, and growth was continued for 2 h. Cells were harvested, washed twice in M63 salt medium (18), and adjusted to to an optical density at 650 nm of 1.2. Transport assays were essentially performed as described previously (19), with the following modifications; 20 l of [ 14 C]maltose (44 kBq) was added to 980 l of cells to a final concentration of 0.5 mM, and 0.3-ml samples were filtered after 1, 5, and 10 min. The high cell density and a high concentration of [ 14 C]maltose were necessary due to the lack of the lamB gene, encoding maltoporin, in strain ED169.…”

“…SDS-PAGE and immunoblotting were performed as described in Ref. 19. Immunoblots were developed using the Western Blot Chemiluminescence Reagent Plus system (NEN Life Science Products).…”