Genetic dissection of the outer membrane secretin PulD: are there distinct domains for multimerization and secretion specificity?
Linker and deletion mutagenesis and gene fusions were used to probe the possible domain structure of the dodecameric outer membrane secretin PulD from the pullulanase secretion pathway of Klebsiella oxytoca. Insertions of 24 amino acids close to or within strongly predicted and highly conserved amphipathic beta strands in the C-terminal half of the polypeptide (the beta domain) abolished sodium dodecyl sulfate (SDS)-resistant multimer formation that is characteristic of this protein, whereas insertions elsewhere generally had less dramatic effects on multimer formation. However, the beta domain alone did not form SDS-resistant multimers unless part of the N-terminal region of the protein (the N domain) was produced in trans. All of the insertions except one, close to the C terminus of the protein, abolished function. The N domain alone was highly unstable and did not form SDS-resistant multimers even when the beta domain was present in trans. We conclude that the beta domain is a major determinant of multimer stability and that the N domain contributes to multimer formation. The entire or part of the N domain of PulD could be replaced by the corresponding region of the OutD secretin from the pectate lyase secretion pathway of Erwinia chrysanthemi without abolishing pullulanase secretion. This suggests that the N domain of PulD is not involved in substrate recognition, contrary to the role proposed for the N domain of OutD, which binds specifically to pectate lyase secreted by E. chrysanthemi (V. E. Shevchik, J. Robert-Badouy, and G. Condemine, EMBO J. 16:3007-3016, 1997).
Guilvout
I
I
Unité de Génétique Moléculaire, CNRS URA 1773, Institut Pasteur, 75724 Paris, France.
Hardie
K R
KR
Sauvonnet
N
N
Pugsley
A P
AP
D019677:Artificial Gene Fusion; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002846:Chromatography, Affinity; D016974:Dickeya chrysanthemi / Q000235:genetics; D017353:Gene Deletion; D015151:Immunoblotting; D007709:Klebsiella / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D016254:Mutagenesis, Insertional; D010641:Phenotype; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D013696:Temperature
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D008565:Membrane Proteins; C106887:OutD protein, Erwinia chrysanthemi; C061378:PulC protein, Klebsiella pneumoniae; C061377:PulD protein, Klebsiella pneumoniae