(126f) Use of a pH Auxostat to Investigate Physiological Responses of E. coli to Rapid Growth Conditions

Microbial cells must adapt to rapidly changing culture environments by alteration of gene expression. While regulatory mechanisms in E. coli have been studied extensively, many questions still remain unanswered. In particular, understanding how growth rate is regulated is critically important for effective use of microorganisms for biotechnology applications. We have explored the cellular response of E. coli to alterations in growth rate using a pH auxostat. An auxostat is a continuous culture system, with feedback control. In the pH auxostat, a pH set point is established slightly lower than the pH of the feed medium (for an acid-producing medium such as a glucose feed). As the cells grow and metabolize the feed, they produce organic acids (e.g. acetate), driving down the pH. To increase the pH, the feed rate is automatically increased. As the feed rate approaches the maximum growth rate of the organism, the culture begins to “wash out”, resulting in less acid production and a reduction in feed rate. Thus, over time, the culture reaches a steady state based on the maximum growth rate of the organism.

Using the pH auxostat, we obtained a specific growth rate of 0.044 min^-1 (corresponding to a doubling time of 15 minutes) for E. coli grown in a minimal medium with 10g/L of glucose and 2 g/L of yeast extract. To determine the changes in gene expression we performed a transcriptomic study using Affymetrix Gene chip arrays. We identified a number of proteins in the RpoS regulon that were differentially expressed. In addition, we found a significant modulation of iron transporter genes. Upregulation of the RpoS system was confirmed by western blotting and increased iron transport was verified by atomic absorption analysis and siderophore analysis. Current studies are focused on identifying the upstream regulators of RpoS in this system and a detailed proteomic analysis of cellular responses. We are also investigating whether the upregulation of RpoS is caused by mutation or simply alteration of gene expression patterns.