Pleiotropic effects of a mutation in rfaC on Escherichia coli hemolysin

Several genes involved in the lipopolysaccharide (LPS) biosynthetic pathway have been shown to affect the expression or activity of Escherichia coli hemolysin (Hly), a secreted cytotoxin that is the prototype of the RTX family of toxins. To further study this relationship, E. coli K-12 strains harboring mutations in the LPS biosynthetic genes rfaS, rfaQ, rfaJ, rfaP, and rfaC were transformed with a recombinant plasmid harboring the hlyCABD operon and examined for their effects on extracellular expression and hemolytic activity. A mutation in rfaC that affected both extracellular expression and activity of Hly was studied in greater detail. This mutation led to a growth-phase-dependent decrease up to 16-fold in the steady-state level of extracellular HlyA, although transcription and secretion of HlyA were decreased no more than 2-fold. Specific hemolytic activity in toxin produced from the rfaC mutant strain was significantly reduced, in a growth-phase-dependent manner. With the rfaC gene supplied in trans, both the decreased expression and activity of Hly were restored to wild-type levels. Hly from the rfaC mutant strain exhibited much slower kinetics of hemolysis, a more rapid rate of decay of activity, and greater formation of apparently inactive HlyA-containing aggregates in culture supernatants than was exhibited in the wild-type strain. A model is proposed for a physical interaction between LPS and Hly in which LPS with intact inner core participates in forming or maintaining an active conformation of Hly and helps to protect it from aggregation or degradation.