"In vivo" detection of L-arabinose-binding protein, CRM-negative mutants

Abstract

An "in vivo" assay for the detection of mutants negative to CRM (cross-reacting material) is described. l-Arabinose-negative mutants of Escherichia coli B/r were grown on Casamino Acids-l-arabinose plates to which a 3-ml agar layer, containing antiserum to the l-arabinose-binding protein (ABP), had been applied. After incubation and partial lysis of the clones "in situ," the plates were refrigerated for 36 hr, rinsed of colonial growth with water, and observed for the presence or absence of an immune precipitation. ABP-minus and l-arabinose regulator (araC)-minus mutants do not produce a precipitin reaction. l-Arabinose isomeraseless (EC 5.3.1.4; araA), kinaseless (EC 2.7.1.16; araB), and epimeraseless (EC 5.1.3.a; araD) mutants produce precipitin reactions. Mutants of E. coli B/r generated by treatment of the wild type with ethyl methane sulfonate or ultraviolet irradiation were isolated, tested for l-arabinose uptake, and screened for the presence or absence of ABP by the described assay. The applications of such an assay are discussed.