Oligomerization of Escherichia coli haemolysin (HlyA) is involved in pore formation.
Coexpression of pairs of nonhaemolytic HlyA mutants in the recombination-deficient (recA) strain Escherichia coli HB101 resulted in a partial reconstitution of haemolytic activity, indicating that the mutation in one HlyA molecule can be complemented by the corresponding wild-type sequence in the other mutant HlyA molecule and vice versa. This suggests that two or more HlyA molecules aggregate prior to pore formation. Partial reconstitution of the haemolytic activity was obtained by the combined expression of a nonhaemolytic HlyA derivative containing a deletion of five repeat units in the repeat domain and several nonhaemolytic HlyA mutants affected in the pore-forming hydrophobic region. The simultaneous expression of two inactive mutant HlyA proteins affected in the region at which HlyA is covalently modified by HlyC and the repeat domain, respectively, resulted in a haemolytic phenotype on blood agar plates comparable to that of wild-type haemolysin. However, complementation was not possible between pairs of HlyA molecules containing site-directed mutations in the hydrophobic region and the modification region, respectively. In addition, no complementation was observed between HlyA mutants with specific mutations at different sites of the same functional domain, i.e. within the hydrophobic region, the modification region or the repeat domain. The aggregation of the HlyA molecules appears to take place after secretion, since no extracellular haemolytic activity was detected when a truncated but active HlyA lacking the C-terminal secretion sequence was expressed together with a nonhaemolytic but transport-competent HlyA mutant containing a deletion in the repeat domain.
Ludwig
A
A
Lehrstuhl für Mikrobiologie, Theodor-Boveri-Institut der Universität Würzburg, Germany.
Benz
R
R
Goebel
W
W
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001704:Biopolymers; D004926:Escherichia coli / Q000378:metabolism* / Q000502:physiology; D029968:Escherichia coli Proteins; D015870:Gene Expression; D005816:Genetic Complementation Test; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism*; D006461:Hemolysis; D016297:Mutagenesis, Site-Directed
D001426:Bacterial Proteins; D001704:Biopolymers; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli