Induction of E. coli recA protein via recBC and alternate pathways: quantitation by enzyme-linked immunosorbent assay (ELISA).
An enzyme-linked immunosorbent assay (ELISA) has been adapted to measure E. coli recA protein in the 1 to 10 ng range in whole-cell sonicates, membrane extracts, and osmotic shock fluid from 2 x 10(8) cells. The specific activity of recA protein is maintained at a relatively constant "basal' level (800 to 1,200 molecules per cell for wild-type E. coli in L-broth, salt-depleted broth and minimal media) during early-log and mid-log phase growth, but it increases by two- to ten-fold as the culture approaches saturation density. Nalidixate-induced levels are 20- to 50-fold higher, and 100-fold higher in a constitutive tif- spr- mutant. Induction of recA protein synthesis by nalidixic acid, which normally requires functional recBC enzyme, also occurs in recB- and recC- cells by pathways activated by mutation in the sbcA and sbcB indirect suppressors. In recB- sbcA- mutants, exonuclease VIII, the recE gene product, is required for induction of recA protein. Abolition of exonuclease I activity by mutation in sbcB allows induction of recA protein by nalidixate in recB- and recC- cells. Mutation in recF does not affect induction by nalidixate in RecBC+ cells, but it enables induction to occur in RecBC- cells, suggesting that recF gene product is involved in regulation of recA protein.
D001426:Bacterial Proteins / Q000096:biosynthesis*; D003851:Deoxyribonucleases / Q000378:metabolism; D004790:Enzyme Induction / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D043211:Exodeoxyribonuclease V; D005092:Exonucleases / Q000378:metabolism; D009154:Mutation; D009268:Nalidixic Acid / Q000494:pharmacology; D011938:Rec A Recombinases; D011995:Recombination, Genetic
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D009268:Nalidixic Acid; D011938:Rec A Recombinases; D003851:Deoxyribonucleases; D005092:Exonucleases; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli