Export and Regulatory Properties of MalE Hybrid Proteins in <i>Escherichia coli</i>

Abstract

The maltose binding protein (MalE) of Escherichia coli is a secreted 370 residue polypeptide which serves as the periplasmic receptor for high-affinity membrane transport of maltose and maltodextrins. Cells tolerate synthesis and translocation across the plasma membrane of large quantities of MalE. Similarly, the outer membrane protein Omp A is not toxic when expressed in physiological amounts. Expression of an ompA-malE hybrid gene consisting of the first 274 residues of OmpA and the last 251 residues of MalE was, however, toxic, although synthesis of the OmpA part alone was not. To examine the effect of synthesis of the MalE part of the hybrid, the malE fragment was cloned into a vector where it was preceded by the gene coding for the OmpA signal peptide and was inducible by isopropy Ithiogalactoside. Upon induction, a polypeptide of the expected size was made, but expression of the truncated MalE turned out to be even more toxic than that of the OmpA-MalE hybrid. The MalE fragment was exported to the periplasm and did not interfere with the export of other secreted polypeptides. A search for suppressors of this toxicity was performed. In a chromosomal gene bank of E. coli, such a suppressor was found which coded for a 102 A-terminal residue fragment of the 217 residue protein NlpE (new outer membrane lipoprotein). NlpE is known to combat the toxicity of the cytosolic ß-galactosidase (LacZ) when artificially exported to the periplasm. This suppression is achieved by activation of the Cpx two-component signal transduction pathway controlling expression of the periplasmic protease DegP. Increased synthesis of DegP caused degradation of LacZ. The same mechanism appears to operate for the degradation of the MalE fragment. In a degP background, the NlpE fragment could not suppress toxicity. Mutants (cpxA*) exist which, without any signal, produced increased levels of DegP. In a cpxA* strain, the MalE fragment was no longer toxic. The toxicity caused by the MalE fragment can still not entirely be explained. However, several lines of evidence, such as the expression of the periplasmic spheroplast protein Y in cells producing MalE251, suggest that the most likely explanation for the lethality is a defect in the cell wall of induced cells