Plaque color method for rapid isolation of novel recA mutants of Escherichia coli K-12: New classes of protease-constitutive recA mutants

Abstract

As a prerequisite to mutational analysis of functional sites on the RecA protein of Escherichia coli, a method was developed for rapid isolation of recA mutants with altered RecA protease function. The method involves plating mutagenized λ recA+ cI ind on strains deleted for recA and containing, as indicators of RecA protease activity, Mu d(Ap lac) fusions in RecA-inducible genes. The λ recA phages were recognized by their altered plaque colors, and the RecA protease activity of the λ recA mutant lysogens was measured by expression of β-galactosidase from dinD::lac. One class of recA mutants had constitutive protease activity and was designated Prt(c); in these cells the RecA protein was always in the protease form without the usual need for DNA damage to activate it. Some Prt(c) mutants were recombinase negative and were designated Prt(c) Rec-. Another class of 65 recA mutants isolated as being protease defective were all also recombinase defective. Unlike the original temperature-dependent Prt(c) Rec+ mutant (recA441), the new Prt(c) Rec+ mutants showed constitutive protease activity at any growth temperature, with some having considerably greater activity than the recA441 strain. Study of these strong Prt(c) Rec+ mutants revealed a new SOS phenomenon, increased permeability to drugs. Use of this new SOS phenomenon as an index of protease strength clearly distinguished 5 Prt(c) mutants as the strongest among 150. These five strongest Prt(c) mutants showed the greatest increase in spontaneous mutation frequency and were not inhibited by cytidine plus guanosine, which inhibited the constitutive protease activity of the recA441 strain and of all the other new Prt(c) mutants. Strong Prt(c) Rec+ mutants were more UV resistant than recA+ strains and showed indications of having RecA proteins whose specific activity of recombinase function was higher than that of wild-type RecA. A Prt+ Rec- mutant with an anomalous response to effectors is described.