Carbodiimide-resistant Membrane Adenosine Triphosphatase in Mutants of Streptococcus faecalis

Abstract

We have isolated mutants of Streptococcus faecalis whose growth is resistant to inhibition by dicyclohexylcarbodiimide (DCCD). These mutants were isolated by direct selection without using a mutagenic agent. It was found that the adenosine triphosphatase in plasma membrane preparations from all the mutant strains was about 100-fold less sensitive to inhibition by DCCD than the enzyme in wild type membranes. In addition the energized uptake of K+ and cycloleucine which is inhibited by DCCD in the wild type organism became resistant to DCCD inhibition in the mutant strain SF-dcc-8. These findings provide genetic evidence for the view that energized uptake of K+ and cycloleucine is coupled to the activity of the membrane ATPase. Sensitivity to DCCD requires that the ATPase be attached to the membrane. To define the site of mutation we studied the DCCD sensitivity of hybrid ATPase-nectin-membrane complexes reconstituted in vitro from isolated wild type and mutant components. When the complex was reconstituted with mutant ATPase, mutant nectin, and wild type depleted membranes, the enzyme was normally sensitive to DCCD. However, when mutant membranes were used for reconstitution the attached ATPase was insensitive to DCCD. These results indicate that in the mutant there is no alteration in either the ATPase or in nectin, the protein required to attach the enzyme to the membrane. It was concluded that resistance of the membrane ATPase to DCCD was due to a defective carbodiimide-sensitizing factor, the membrane component that mediates DCCD inhibition.