Making metabolism accessible and meaningful: is the definition of a central metabolic dogma within reach?

Intermediary metabolism, a dominant research area before the emergence of molecular biology, is attracting renewed interest for fundamental and applied reasons as documented here. Nonetheless, the field may appear to be a thicket precluding entry to all but the most determined. Here we present a metabolic overview that makes this important and fascinating area accessible to a broad range of the molecular biological and biotechnological communities that are being attracted to biological problems crying out for metabolic solutions.

Bioluminescent Escherichia coli strains for the quantitative detection of phosphate and ammonia in coastal and suburban watersheds.

Accumulation of phosphate and ammonia in estuarine systems and subsequent dinoflagellate and algal blooms has been implicated in fish kills and in health risks for fishermen. Analytic chemistry kits are used to measure phosphate and ammonia levels in water samples, but their sensitivity is limited due to specificity for inorganic forms of these moieties. An Escherichia coli bioluminescent reporter system measured the bioavailability of inorganic nutrients through fusion of E.

Chromosomal promoter replacement of the isoprenoid pathway for enhancing carotenoid production in E. coli.

For metabolic engineering it is advantageous in terms of stability, genetic regulation, and metabolic burden to modulate expression of relevant genes on the chromosome rather than relying on over-expression of the genes on multi-copy vectors. Here we have increased the production of beta-carotene in Escherichia coli by replacing the native promoter of the chromosomal isoprenoid genes with the strong bacteriophage T5 promoter (P(T5)). We recombined PCR fragments with the lambda-Red recombinase to effect chromosomal promoter replacement, which allows direct integration of a promoter along with a selectable marker that can subsequently be excised by the Flp/FRT site-specific recombination system.

Prominent roles of the NorR and Fur regulators in the Escherichia coli transcriptional response to reactive nitrogen species.

We examined the genomewide transcriptional responses of Escherichia coli treated with nitrosylated glutathione or the nitric oxide (NO)-generator acidified sodium nitrite (NaNO(2)) during aerobic growth. These assays showed that NorR, a homolog of NO-responsive transcription factors in Ralstonia eutrophus, and Fur, the global repressor of ferric ion uptake, are major regulators of the response to reactive nitrogen species. In contrast, SoxR and OxyR, regulators of the E.

Interfering with different steps of protein synthesis explored by transcriptional profiling of Escherichia coli K-12.

Escherichia coli responses to four inhibitors that interfere with translation were monitored at the transcriptional level. A DNA microarray method provided a comprehensive view of changes in mRNA levels after exposure to these agents. Real-time reverse transcriptase PCRanalysis served to verify observations made with microarrays, and a chromosomal grpE::lux operon fusion was employed to specifically monitor the heat shock response.

Uses and pitfalls of microarrays for studying transcriptional regulation.

Microarrays provide a powerful new tool for understanding the regulation of gene expression in bacteria. Many recent publications have used microarrays for identifying regulon members and stimulons that describe the complex organismal responses to environmental perturbations. The use of bioinformatics to identify DNA binding sites of transcription factors greatly facilitates the interpretation of these experiments.

Gene expression analysis of the response by Escherichia coli to seawater.

Gene expression of Escherichia coli cells exposed to seawater for 20 h was compared to that of exponentially growing cells (mops-glucose 0.2%) using DNA microarray technology. The expression of most (ca.

Global gene expression profiles of the cyanobacterium Synechocystis sp. strain PCC 6803 in response to irradiation with UV-B and white light.

We developed a transcript profiling methodology to elucidate expression patterns of the cyanobacterium Synechocystis sp. strain PCC 6803 and used the technology to investigate changes in gene expression caused by irradiation with either intermediate-wavelength UV light (UV-B) or high-intensity white light. Several families of transcripts were altered by UV-B treatment, including mRNAs specifying proteins involved in light harvesting, photosynthesis, photoprotection, and the heat shock response.

Impact of genomic technologies on studies of bacterial gene expression.

The ability to simultaneously monitor expression of all genes in any bacterium whose genome has been sequenced has only recently become available. This requires not only careful experimentation but also that voluminous data be organized and interpreted. Here we review the emerging technologies that are impacting the study of bacterial global regulatory mechanisms with a view toward discussing both perceived best practices and the current state of the art.

Recombinant microorganisms as environmental biosensors: pollutants detection by Escherichia coli bearing fabA'::lux fusions.

A set of genetically engineered Escherichia coli strains was constructed, in which the promoter of the fabA gene is fused to Vibrio fischeri luxCDABE either in a multi-copy plasmid or as a single copy chromosomal integration. The fabA gene codes for beta-hydroxydecanoyl-ACP dehydrase, a key enzyme in the synthesis of unsaturated fatty acids, and is induced when fatty acid biosynthesis pathways are interrupted. A dose-dependent and highly sensitive bioluminescent response to a variety of chemicals was controlled by the fadR gene.