Global Expression Profiling of Acetate-grown Escherichia coli

Abstract

This study characterized the transcript profile of Escherichia coli in acetate cultures using DNA microarray on glass slides. Glucose-grown cultures were used as a reference. At the 95% confidence level, 354 genes were up-regulated in acetate, while 370 genes were downregulated compared with the glucose-grown culture. Generally, more metabolic genes were up-regulated in acetate than other gene groups, while genes involved in cell replication, transcription, and translation machinery tended to be down-regulated. It appears that E. coli commits more resources to metabolism at the expense of growth when cultured in the poor carbon source. The expression profile confirms many known features in acetate metabolism such as the induction of the glyoxylate pathway, tricarboxylic acid cycle, and gluconeogenic genes. It also provided many previously unknown features, including induction of malic enzymes, ppsA, and the glycolate pathway and repression of glycolytic and glucose phosphotransferase genes in acetate. The carbon flux delivered from the malic enzymes and PpsA in acetate was further confirmed by deletion mutations. In general, the gene expression profiles qualitatively agree with the metabolic flux changes and may serve as a predictor for gene function and metabolic flux distribution.Physiological characteristics of Escherichia coli using acetate or glucose as a sole carbon and energy source have been studied for more than three decades (1, 2). Briefly, E. coli uptakes glucose using the phosphotransferase system that converts extracellular glucose into intracellular glucose 6-phosphate, which can be further metabolized by the glycolytic pathway to produce energy and biosynthetic precursors. In the presence of glucose, the adenylate cyclase is inactive, and the cAMP level is low. In the absence of glucose, the adenylate cyclase is activated to produce cAMP, which when binding to the cAMP receptor protein activates the expression of a large set of catabolite derepression genes (2, 3). On the other hand, acetate is transported into the cell and converted to acetyl-CoA, which is further metabolized through the glyoxylate shunt and the tricarboxylic acid cycle. The acetate-metabolizing genes are typically repressed in the presence of glucose. The induction and regulation of acetate-metabolizing genes have been studied extensively (4). Since the two carbon sources, glucose and acetate, are utilized by distinct metabolic pathways, the metabolic flux distribution differs significantly in these two carbon sources (5). Understanding global gene expression profiles in different carbon sources is important to the investigation of E. coli growth in natural environment, where the availability of carbon sources changes dynamically. Acetate-metabolizing culture is particularly relevant to the biotechnology industry, since the accumulation of acetate in bioreactor is commonly observed and often poses as an obstacle to high cell density cultivation (6).The recent advent of microarray technology allows a thorough analysis of gene ex...

Transcriptome-based determination of multiple transcription regulator activities in Escherichia coli by using network component analysis

“…DNA microarray has been successfully used as a tool in the study of differential gene expression changes in various conditions (6,(18)(19)(20)(21)(22)(23)(24). However, without the proper mathematical and statis- tical tools, it is difficult to decipher the complex physiological responses to these environmental perturbations from these gene expression studies (25).…”

“…Total RNA of Ϸ1 ϫ 10 9 cells was purified by hot phenol extraction followed by purification using the RNeasy midi kit (Qiagen) following the manufacturer's instructions. cDNA labeling and hybridization were performed according to protocols as described (6). DNA microarray slides containing all E. coli ORFs were made in our laboratory (6), and the images were scanned by using a Versarray ChipReader high-resolution (5 m) scanner (Bio-Rad) at two excitation wavelengths (532 and 635 nm).…”

“…cDNA labeling and hybridization were performed according to protocols as described (6). DNA microarray slides containing all E. coli ORFs were made in our laboratory (6), and the images were scanned by using a Versarray ChipReader high-resolution (5 m) scanner (Bio-Rad) at two excitation wavelengths (532 and 635 nm). Each slide contained two spots of the same probe, each sample was hybridized to two slides, and the time-course experiments were replicated up to three times.…”

“…When switching from a glycolytic condition to a gluconeogenic condition with acetate as the sole carbon source, E. coli is known to induce a significant change in metabolic genes (5). In particular, the glyoxylate shunt (ace-BAK), the tricarboxylic acid (TCA) cycle, and the acetate uptake gene (acs) are up-regulated along with other genes under catabolite repression (6). The gene expression pattern has been studied by comparing the balanced growth culture in glucose and acetate media (6).…”

“…In particular, the glyoxylate shunt (ace-BAK), the tricarboxylic acid (TCA) cycle, and the acetate uptake gene (acs) are up-regulated along with other genes under catabolite repression (6). The gene expression pattern has been studied by comparing the balanced growth culture in glucose and acetate media (6). However, how the cell coordinates the transition from one condition to the other during the adaptation phase has not been characterized.…”