Genetic Analysis of Components Involved in Vitamin B 12 Uptake in Escherichia coli

Abstract

The products of three genes are involved in cyanocobalamin (B 12 ) uptake in Escherichia coli. btuB (formerly bfe ), located at min 88 on the Escherichia coli linkage map, codes for a protein component of the outer membrane which serves as receptor for B 12 , the E colicins, and bacteriophage BF23. Four phenotypic classes of mutants varying in response to these agents were found to carry mutations that, based on complementation and reversion analyses, reside in the single btuB cistron. In one mutant class, ligand binding to the receptor appeared to be normal, but subsequent B 12 uptake was defective. The level of receptor and rate of uptake were responsive to btuB gene dosage. Previous studies showed that the tonB product was necessary for energy-dependent B 12 uptake but not for its binding. Other than those in tonB , no mutations that conferred insensitivity to group B colicins affected B 12 utilization. The requirement for the btuB and tonB products could be bypassed by elevated levels of B 12 (>1 μM) or by mutations compromising the integrity of the outer membrane as a permeability barrier. Utilization of elevated B 12 concentrations in strains lacking the btuB-tonB uptake system was dependent on the function of the btuC product. This gene was located at 37.7 min on the linkage map, with the order pps-btuC-pheS . Strains altered in btuC but with an intact btuB-tonB system were only slightly impaired in B 12 utilization, being defective in its accumulation. This defect was manifested as inability to retain B 12 , such that intracellular label was almost completely lost by exchange or efflux. It is proposed that btuC encodes a transport system for B 12 in the periplasm.