Characterization of Gene Expression of the E. coli abg Region, Which Encodes Proteins Involved in Folic Acid Catabolism
Authors: Patel Niytai , Pitstick Lenore , Green Jacalyn 
Published: November 2023

Introduction

Folic acid is a water‐soluble vitamin that is essential for many cellular processes such as biosynthesis of DNA, RNA, and amino acids. In E. coli, the abg region encodes proteins that enable the uptake and cleavage of p‐aminobenzoyl‐glutamate (PABA‐GLU), a breakdown product of folic acid. These genes comprise an apparent operon oriented divergently from abgR, encoding a putative transcriptional regulator. Since this operon is only found in bacterial chromosomes, understanding its cellular role may provide new drug targets. Additionally, it has been noted in some studies that changes in the abgR region are characteristic of some pathogenic strains of E. coli. The goal of this study was to characterize expression and regulation of the abg operon. Our hypothesis was that AbgR regulated expression of the divergently transcribed abgABT genes.

Methods

E coli strains studied included wild type Keio strain BW25113, JW1333 (abgR::kan) and a strain with a point mutation in the intergenic region between abgR and abgA (BN1140). Cells were grown in Luria Broth (LB) and in Vogel Bonner (VB) glucose minimal medium with and without supplemental PABA‐GLU (100 μM). Cell growth was monitored by measuring absorbance at 600 nm. Cells were harvested at several points in the growth curve: ~0.2 OD600, ~0.5 OD600, and in early stationary phase (~1.0 OD600). RNA was isolated using a RiboPure‐Bacteria kit (Ambion, Austin, TX). Primers were designed for abgR, abgA, abgB, and abgT. Primers were tested for dynamic range and efficiency. Quantitative real time PCR was performed using Power SYBR Green from Applied Biosystems (Carlsbad, CA) and the 7300 Real Time PCR Applied Biosystems instrument. Standard curves were used to determine the absolute transcript level for each gene. Data were analyzed using GraphPad Prism 5.

Results

In JW1333 cells grown in LB to stationary phase, mRNA levels of abgA, abgB, and abgT were higher (7.0‐fold, 2.3‐fold, and 2.3‐fold, respectively) when compared to expression in BW25113; this is consistent with AbgR functioning as a transcriptional repressor. When comparing expression of these four genes in BW25113 cells grown in LB or minimal medium, harvested at 0.2 OD, 0.5 OD, and 1.0 OD, results varied depending on the gene. AbgR was expressed more highly at every condition than the abgABT genes. At 0.2 OD, abgA, abgB and abgT were more highly expressed in minimal medium (8.6‐, 5.3‐ and 6.2 fold respectively) than in LB. In cells harvested at 0.5 OD and 1.0 OD, expression varied much less for each growth condition, between 0.8 and 2.5‐fold. BN1140 expressed 9–30‐fold more copies of AbgABT than wildtype (BW25113) cells; little change in AbgR expression was observed. Thus, the point mutation in the intergenic region affected abgABT expression alone. Wild type cells expressing a high‐copy plasmid encoding abgT, enabling import of PABA‐GLU, and grown in the absence and presence of 100 μM PABA‐GLU, demonstrated no differences in expression. This showed that expression of this region is independent of the cellular concentration of PABA‐GLU.

Conclusion

AbgR acts as a transcriptional repressor of divergently transcribed abgA, abgB and abgT. Expression of the abg region is dependent on nutrient availability. PABA‐GLU does not control the expression of the abg operon.

Support or Funding Information

Acknowledgements: This work was supported by Midwestern University.