A novel HPLC based approach to characterizing and quantitating nucleotide pools in bacteria.

摘要

Nucleotide pools, in addition to being important metabolites in the cell, can have very important effects on cellular physiology. The relative concentrations of nucleotides can affect processes in bacteria ranging from determining the growth rate to sporulation to gene transcription. In the past, nucleotide pools were measured via radioactive labeling and two-dimensional thin layer chromatography or by manipulation of large cultures for analysis on HPLC. This work developed a novel approach for characterizing and quantitating the nucleotide pools by combining the pool-preservation of formic acid extraction with the quantitation of HPLC. In developing the methodology, this work exposes the tremendous lability of the nucleotide pools, especially ATP. Even minor manipulations of cells, such as letting them settle in nutrient rich media for 2 minutes, can result in a significant reduction in the ATP pool size and induction of a stringent response. The final method, which involves mixing formic acid into growing media and then collecting the extracted nucleotides on a Q-sepharose column followed by dialysis and lyophilization, accurately preserves the pools for quantification.; The new method was also used to answer some outstanding questions in the literature and expose novel biology. Using this method, it was determined that the ATP pools do not change with growth rate and remain constant above 3mM. Unless there is significant pool sequestration, this means that the concentration of ATP is well above the KATP of most promoters and enzymes in the cell. The new method was also used to track the nucleotide pools as E. coli passes from log-phase into stationary phase. Distinct patterns of nucleotide pools were observed with most nucleotides dropping sharply as the cells transition into stationary phase followed by a rebound in concentration and then a general decrease. Some nucleotide species, of note UTP and UDP-glucose/galactose, surprisingly increased as the cells enter into stationary phase. This method thus serves as a new tool to answer questions and expose new phenomenon involving nucleotide biochemistry in bacteria.