Metabolism of Radioactive Para-Aminobenzoic Acid in an Escherichia coli Mutant.

Abstract

While metabolism of para-aminobenzoate (P-AB) has been extensively investigated in the mammalian body( 1,2), its function and biochemical transformations in microorganisms are still obscure. Conversion of PAB into folic acid and the citrovorum factor occurs in some organisms (3,4) a1 though there is some question whether these are the active forms of PAB. A number of other routes of metabolism have been reported. Davis( 5 ,6 ) has implicated PAB in the metabolism by Escherichia C O Z~ of tryptophane, tyrosine and other aromatic compounds, as well as vit. BI2. Ratner et aZ.( 7 ) isolated a glutamic acid peptide of PAB from yeast, and Flynn et d ( 8 ) have found in 2 strains of streptococci an antibiotic containing PAB and cytosine among its components. Sloane et a2. (9) identified aniline and p-aminophenol as degradation products in Mycobacterium smegmatis. The present studies had their inception in the isolation by Lampen, Roepke, and Jones(l0) of an X-ray mutant of E. coZi which required exogenous PAB for growth but apparently did *Supported in part by grant from U. S. Atomic Energy Commission, Contract S o . .4T(30-1) -1107 and the C. S. -4ir Force, Contract No. -4F 18(600) -853. t Portions of these data were used in partial satisfaction of requirements for degree of Master of Science, George Washington University.not utilize it in producing folic acid. Since the wild strain of this organism normally has no requirement for preformed PAB. it seemed od interest to investigate the fate of this compound labeled with C14 in such a mutant.Materials and methods. PAB used was labeled in the carboxyl group with carbon 14 and had a specific activity of 0.037 mC/mg.$ The mutant of E . coli (ATCC S o . 9723a) was grown on basal medium modified from that of Cohen and Arbogast( 11) by use of 0.4% glucose. The medium of Lampen et aZ.( 10) contained asparagine as well, but this substance was not necessary for maximum growth, and was omitted to facilitate isolations. Organisms were grown in 3 liter batches with aeration for 24 hours at PAB concentration of 0.01 &ml.Experiments in which PAB level was 0.05 &ml gave similar results. Bacterial suspensions were filtered on Buechner funnel using Celite as filter aid. The filtrate contained less than 207% of total radioactivity. The cells, plus filster-aid, were suspended in water and made up to 60% acetone-water (v/v) by addition of acetone. The residue was filtered off, re-extracted, and extracts, containing approximately half the total cell radioactivity, were combined. Subsequent extractions of cells with hot acetone. chloroform, $ Purchased from Atomic Energy 'Commission, Los Alamos Laboratories.

RELATIONSHIP BETWEEN PURINES AND FOLIC ACID-VITAMINS IN THE MICROCOCCUS, GAFFKYA HOMARI

“…Other workers have found that leucovorin is a stable form of metabolically active folic acid when cells or cellular homogenates are either grown or incubated with p-aminobenzoic or pteroylglutamic acid. Reid and Couch (1955) found that chicken liver homogenates converted pteroylglutamic acid to leucovorin; Nichol (1954) reported that cellfree extracts of S. faecalis converted pteroylglutamic acid to leucovorin; Laseelles and Woods (1954) reported that suspensions of S. faecalis strain ATCC 8043 incubated with pteroylglutamic acid produce leucovorin; Davidson et al (1956) fed radioactive p-aminobenzoic acid to a p-aminobenzoic acid-requiring mutant of E. coli and isolated radioactive compounds which seemed to resemble leucovorin.…”