Secretion and activation of the Serratia marcescens hemolysin by structurally defined ShlB mutants.
The ShlA hemolysin of Serratia marcescens is secreted across the outer membrane by the ShlB protein; ShlB belongs to the two-partner secretion system (type Vb), a subfamily of the Omp85 outer membrane protein assembly and secretion superfamily. During secretion, ShlA is converted from an inactive non-hemolytic form into an active hemolytic form. The structure of ShlB is predicted to consist of the N-terminal α-helix H1, followed by the two polypeptide-transport-associated domains POTRA P1 and P2, and the β-barrel of 16 β-strands. H1 is inserted into the pore of the β-barrel in the outer membrane; P1 and P2 are located in the periplasm. To obtain insights into the secretion and activation of ShlA by ShlB, we isolated ShlB mutants impaired in secretion and/or activation. The triple H1 P1 P2 mutant did not secrete ShlA. The P1 and P2 deletion derivatives secreted reduced amounts of ShlA, of which P1 showed some hemolysis, whereas P2 was inactive. Deletion of loop 6 (L6), which is conserved among exporters of the Omp85 family, compromised activation but retained low secretion. Secretion-negative mutants generated by random mutagenesis were located in loop 6. The inactive secreted ShlA derivatives were complemented in vitro to active ShlA by an N-terminal ShlA fragment (ShlA242) secreted by ShlB. Deletion of H1 did not impair secretion of hemolytic ShlA. The study defines domains of ShlB which are important for ShlA secretion and activation.
Max Planck Institute for Developmental Biology, Department of Protein Evolution, Spemannstrasse 35, 72076 Tübingen, Germany.
Avijit
A
Pramanik
Max Planck Institute for Developmental Biology, Department of Protein Evolution, Spemannstrasse 35, 72076 Tübingen, Germany.
Ulrich
U
Könninger
Max Planck Institute for Developmental Biology, Department of Protein Evolution, Spemannstrasse 35, 72076 Tübingen, Germany.
Arun
A
Selvam
Max Planck Institute for Developmental Biology, Department of Protein Evolution, Spemannstrasse 35, 72076 Tübingen, Germany. Electronic address: volkmar.braun@tuebingen.mpg.de.
Volkmar
V
Braun
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005816:Genetic Complementation Test; D006460:Hemolysin Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011487:Protein Conformation; D017384:Sequence Deletion; D012706:Serratia marcescens / Q000235:genetics* / Q000378:metabolism*
D001426:Bacterial Proteins; D006460:Hemolysin Proteins; D050505:Mutant Proteins; C089911:ShlA protein, Serratia marcescens; C107896:ShlB protein, Serratia marcescens
Activation; Hemolysin; Secretion; Serratia marcescens