Escherichia coli abg genes enable uptake and cleavage of the folate catabolite p-aminobenzoyl-glutamate

Abstract

Escherichia coli AbgT was first identified as a structural protein enabling the growth of p-aminobenzoate auxotrophs on exogenous p-aminobenzoyl-glutamate (M. J. Hussein, J. M. Green, and B. P. Nichols, J. Bacteriol. 180:6260-6268, 1998). The abg region includes abgA, abgB, abgT, and ogt; these genes may be regulated by AbgR, a divergently transcribed LysR-type protein. Wild-type cells transformed with a high-copy-number plasmid encoding abgT demonstrate saturable uptake of p-aminobenzoyl-glutamate (K(T)=123 microM); control cells expressing vector demonstrate negligible uptake. The addition of metabolic poisons inhibited uptake of p-aminobenzoyl-glutamate, consistent with this process requiring energy. p-Aminobenzoyl-glutamate taken in by cells expressing large amounts of AbgT alone is not rapidly metabolized to a form that is trapped in the cell, as the addition of nonradioactive p-aminobenzoyl-glutamate to these cells results in a rapid loss of intracellular label. The addition of nonradioactive p-aminobenzoate has no effect. The abgA, abgB, and abgAB genes were cloned into the medium-copy-number plasmid pACYC184; p-aminobenzoate auxotrophs transformed with the clone encoding abgAB demonstrated enhanced ability to grow on low levels of p-aminobenzoyl-glutamate. When transformed with complementary plasmids encoding high-copy levels of abgT and medium-copy levels of abgAB, p-aminobenzoate auxotrophs grew on 50 nM p-aminobenzoyl-glutamate. Our data are consistent with a model of p-aminobenzoyl-glutamate utilization in which AbgT catalyzes transport of p-aminobenzoyl-glutamate, followed by cleavage to p-aminobenzoate by a protein composed of subunits encoded by abgA and abgB. While endogenous expression of these genes is very low under the conditions in which we performed our experiments, these genes may be induced by AbgR bound to an unknown molecule. The true physiological role of this region may be related to some molecule similar to p-aminobenzoyl-glutamate, such as a dipeptide.

FIG. 1.

Structures of folic acid, p-aminobenzoate (PABA), and p-aminobenzoyl-glutamate (pABA-GLU).

FIG. 2.

abg region of the chromosome.

FIG. 3.

Transport of radioactive p-aminobenzoyl-glutamate. (A) Time course of p-aminobenzoyl-glutamate uptake by MG1655 cells transformed with pJMG128 (a pUC18-derived plasmid expressing AbgT) and the parent vector pUC18. Assays were performed as described in Materials and Methods. Briefly, cells were grown overnight, washed, and resuspended in minimal medium. After preincubation, the transport assay (final volume, 6 ml) was initiated by the addition of [³H]p-aminobenzoyl-glutamate to a final concentration of 50 μM. Duplicate samples (0.1 ml) were taken at the times shown following initiation. Samples were filtered, immediately washed with ice-cold minimal medium, and counted. Data were analyzed with GraphPad Prism 4; error bars represent standard errors. (B) Effect of p-aminobenzoyl-glutamate concentration on the initial uptake of [³H]p-aminobenzoyl-glutamate by MG1655 transformed with pJMG128. Assays were performed as described in Materials and Methods. After preincubation, the transport assay (final volume, 6 ml) was initiated by the addition of [³H]p-aminobenzoyl-glutamate; final concentrations used were 12.5 μM, 16.7 μM, 25 μM, 50 μM, 100 μM, 150 μM, and 200 μM. For each concentration, a reaction was initiated and duplicate samples (0.4 ml) were taken within the first few minutes following initiation. Samples were filtered, immediately washed with ice-cold minimal medium, and counted. For each concentration, linear regression analysis was performed, and the slope of the line was measured and taken as the initial velocity. Data were analyzed with GraphPad Prism 4; error bars represent standard errors. (C) Lineweaver-Burk plot of the data from panel B.

FIG. 4.

Growth of p-aminobenzoate auxotrophs (BN1103) transformed with various plasmids encoding genes of the abg region in liquid medium containing various concentrations of p-aminobenzoyl-glutamate. (A) Comparison of the growth of BN1103 transformed with pACYC184, pLJA, pLJB, pLJAB, pUC18, pJG128, and pLJAB/pJG128 in minimal medium containing various concentrations of p-aminobenzoyl-glutamate at 48 h after inoculation. BN1103 (pabA) cells transformed with pACYC184, pLJA, pLJB, pLJAB, pUC18, pJG128, and pLJAB/pJG128 were grown overnight in minimal medium containing the appropriate antibiotics for plasmid selection and 100 nM p-aminobenzoate, washed three times with sterile saline, and diluted 10⁵ into 5 ml of minimal medium containing antibiotic and increasing concentrations of p-aminobenzoyl-glutamate. Cultures were incubated at 37°C; the optical density (OD) (λ = 600 nm) of a 1-ml sample was measured every 24 h. The loss in volume due to evaporation was about 2.5% per day (∼8% loss after 72 h). Results are given as means ± standard errors. (B) Readings of the data from panel A at 72 h.

FIG. 5.

Transport and retention of radioactive p-aminobenzoyl-glutamate. (A) Effect of the addition of unlabeled p-aminobenzoyl-glutamate or p-aminobenzoate on cells (MG1655 transformed with pJMG128) that have accumulated radioactive p-aminobenzoyl-glutamate. Assays were performed as described in Materials and Methods. Briefly, cells were grown overnight, washed, and resuspended in minimal medium. After preincubation, the transport assay (final volume, 6 ml) was initiated by the addition of [³H]p-aminobenzoyl-glutamate to a final concentration of 50 μM. Duplicate samples (0.4 ml) were taken at the times shown. After the 180-min sample was taken, the mixture was divided in two equal portions and a nonradioactive sample of p-aminobenzoyl-glutamate (final concentration, 10 mM) or p-aminobenzoate (final concentration, 2 mM) was added. Duplicate samples were then taken of each mixture at 210 and 240 min. Samples were filtered, immediately washed with ice-cold minimal medium, and counted. Data were analyzed with GraphPad Prism 4; error bars represent standard errors. (B) Transport and retention of [³H]p-aminobenzoyl-glutamate by MG1655 transformed with both pJMG128 and pLJAB. Assays were performed as described in Materials and Methods. After preincubation, the transport assay (final volume, 6 ml) was initiated by the addition of [³H]p-aminobenzoyl-glutamate to a final concentration of 50 μM. Duplicate samples (0.1 ml) were taken at the times shown. Samples were filtered, immediately washed with ice-cold minimal medium, and counted. Data were analyzed with GraphPad Prism 4; error bars represent standard errors.