We consider the problem of comparing the gene expression levels of cells grown under two different conditions using cDNA microarray data. We use a quality index, computed from duplicate spots on the same slide, to filter out outlying spots, poor quality genes and problematical slides. We also perform calibration experiments to show that normalization between fluorescent labels is needed and that the normalization is slide dependent and non-linear. A rank invariant method is suggested to select non-differentially expressed genes and to construct normalization curves in comparative experiments. After normalization the residuals from the calibration data are used to provide prior information on variance components in the analysis of comparative experiments. Based on a hierarchical model that incorporates several levels of variations, a method for assessing the significance of gene effects in comparative experiments is presented. The analysis is demonstrated via two groups of experiments with 125 and 4129 genes, respectively, in Escherichia coli grown in glucose and acetate.

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...

Syntrophic metabolism is diverse in two respects: phylogenetically with microorganisms capable of syntrophic metabolism found in the Deltaproteobacteria and in the low G+C gram-positive bacteria, and metabolically given the wide variety of compounds that can be syntrophically metabolized. The latter includes saturated fatty acids, unsaturated fatty acids, alcohols, and hydrocarbons. Besides residing in freshwater and marine anoxic sediments and soils, microbes capable of syntrophic metabolism also have been observed in more extreme habitats, including acidic soils, alkaline soils, thermal springs, and permanently cold soils, demonstrating that syntrophy is a widely distributed metabolic process in nature. Recent ecological and physiological studies show that syntrophy plays a far larger role in carbon cycling than was previously thought. The availability of the first complete genome sequences for four model microorganisms capable of syntrophic metabolism provides the genetic framework to begin dissecting the biochemistry of the marginal energy economies and interspecies interactions that are characteristic of the syntrophic lifestyle.

Biochemically, the syntrophic bacteria constitute the missing link in our understanding of anaerobic flow of carbon in the biosphere. The completed genome sequence of Syntrophus aciditrophicus SB, a model fatty acid-and aromatic acid-degrading syntrophic bacterium, provides a glimpse of the composition and architecture of the electron transfer and energy-transducing systems needed to exist on marginal energy economies of a syntrophic lifestyle. The genome contains 3,179,300 base pairs and 3,169 genes where 1,618 genes were assigned putative functions. Metabolic reconstruction of the gene inventory revealed that most biosynthetic pathways of a typical Gram-negative microbe were present. A distinctive feature of syntrophic metabolism is the need for reverse electron transport; the presence of a unique Rnf-type ion-translocating electron transfer complex, menaquinone, and membrane-bound Fe-S proteins with associated heterodisulfide reductase domains suggests mechanisms to accomplish this task. Previously undescribed approaches to degrade fatty and aromatic acids, including multiple AMP-forming CoA ligases and acyl-CoA synthetases seem to be present as ways to form and dissipate ion gradients by using a sodium-based energy strategy. Thus, S. aciditrophicus, although nutritionally self-sufficient, seems to be a syntrophic specialist with limited fermentative and respiratory metabolism. Genomic analysis confirms the S. aciditrophicus metabolic and regulatory commitment to a nonconventional mode of life compared with our prevailing understanding of microbiology.anaerobic food chains ͉ syntrophic metabolism ͉ fatty acid and benzoate utilization

SummarySyntrophomonas wolfei is a specialist, evolutionarily adapted for syntrophic growth with methanogens and other hydrogen-and/or formate-using microorganisms. This slow-growing anaerobe has three putative ribosome RNA operons, each of which has 16S rRNA and 23S rRNA genes of different length and multiple 5S rRNA genes. The genome also contains 10 RNAdirected, DNA polymerase genes. Genomic analysis shows that S. wolfei relies solely on the reduction of protons, bicarbonate or unsaturated fatty acids to re-oxidize reduced cofactors. Syntrophomonas wolfei lacks the genes needed for aerobic or anaerobic respiration and has an exceptionally limited ability to create ion gradients. An ATP synthase and a pyrophosphatase were the only systems detected capable of creating an ion gradient. Multiple homologues for b-oxidation genes were present even though S. wolfei uses a limited range of fatty acids from four to eight carbons in length. Syntrophomonas wolfei, other syntrophic metabolizers with completed genomic sequences, and thermophilic anaerobes known to produce high molar ratios of hydrogen from glucose have genes to produce H 2 from NADH by an electron bifurcation mechanism. Comparative genomic analysis also suggests that formate production from NADH may involve electron bifurcation. A membrane-bound, iron-sulfur oxidoreductase found in S. wolfei and Syntrophus aciditrophicus may be uniquely involved in reverse electron transport during syntrophic fatty acid metabolism. The genome sequence of S. wolfei reveals several core reactions that may be characteristic of syntrophic fatty acid metabolism and illustrates how biological systems produce hydrogen from thermodynamically difficult reactions.