| Like many folate synthesizing organisms, E. coli lacks folate transport systems. To isolate folate uptake mutants, a collection of p-aminobenzoate auxotrophs (pabA{dollar}sp-{dollar}) was subjected to diethylsulphonate mutagenesis which resulted in isolation of several clones that grew on folic acid minimal medium. The vigorous growth exhibited by the mutants on folic acid medium was found to be due to a minor contaminant, namely, p-aminobenzoyl glutamic acid, (PABG), a degradation product of folic acid. Growth on PABG required the concerted effect of two gene activities. One activity performs the function of transporting PABG compound intracellularly. The other activity functions to cleave the transported PABG into its components, p-aminobenzoate (PABA) and glutamate. The released PABA is believed to be utilized by the cells to synthesize folic acid. A 5,640 kb gene cluster which is associated with transport and cleavage of PABG was cloned and sequenced. The abg gene cluster contains three similarly oriented structural genes and one divergently oriented regulatory gene. The divergent gene, abgR, is highly homologous to the LysR gene family of transcriptional regulators. Situated downstream from abgR are abgA and abgB, which overlap by one base pair. The two genes are homologous to each other and to a shared set of hydrolases, most likely they code for two polypeptides which associate together to form the PABG cleavage enzyme. The fourth gene in the cluster is abgT, which encodes a highly hydrophobic membrane associated transport protein. The genetic organization suggests that the abg gene cluster might be constituting an operon that is coordinately regulated by abgR. Two types of point mutations were found in the intergenic region between abgR and the rest of the genes in the cluster in four of the six mutants investigated. The other two mutants showed a 4.5 kb chromosomal duplication that included the abg gene cluster or parts of it. |
