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. This is accomplished by identifying seven key concepts, a so-called metabolic central dogma, that provide a core understanding analogous to the "Central Dogma of Molecular Biology" which focused upon maintenance and flow of genetic information.

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. coli promoters (phoA or glnAp2) to the luxCDABE operon of Vibrio fischeri carried either on the chromosome or on a multicopy plasmid vector, resulting in emission of light in response to phosphate or ammonia starvation. Responses were shown to be under the control of expected physiological regulators, phoB and glnFG, respectively. Standard curves were used to determine the phosphate and ammonia levels in water samples from diverse watersheds located in the northeastern United States. Bioluminescence produced in response to nutrient starvation correlated with concentrations of phosphate (1-24 ppm) and ammonia (0.1-1.6 ppm). While the ammonia biosensor measured nutrient concentrations in tested water samples that were comparable to the amounts reported by a commercial kit, the phosphate biosensor reported higher levels of phosphate in Chesapeake water samples than did the kit.

Quality control despite mistranslation caused by an ambiguous genetic code.

A high level of accuracy during protein synthesis is considered essential for life. Aminoacyl-tRNA synthetases (aaRSs) translate the genetic code by ensuring the correct pairing of amino acids with their cognate tRNAs. Because some aaRSs also produce misacylated aminoacyl-tRNA (aa-tRNA) in vivo, we addressed the question of protein quality within the context of missense suppression by Cys-tRNA(Pro), Ser-tRNA(Thr), Glu-tRNA(Gln), and Asp-tRNA(Asn). Suppression of an active-site missense mutation leads to a mixture of inactive mutant protein (from translation with correctly acylated aa-tRNA) and active enzyme indistinguishable from the wild-type protein (from translation with misacylated aa-tRNA). Here, we provide genetic and biochemical evidence that under selective pressure, Escherichia coli not only tolerates the presence of misacylated aa-tRNA, but can even require it for growth. Furthermore, by using mass spectrometry of a reporter protein not subject to selection, we show that E. coli can survive the ambiguous genetic code imposed by misacylated aa-tRNA tolerating up to 10% of mismade protein. The editing function of aaRSs to hydrolyze misacylated aa-tRNA is not essential for survival, and the EF-Tu barrier against misacylated aa-tRNA is not absolute. Rather, E. coli copes with mistranslation by triggering the heat shock response that stimulates nonoptimized polypeptides to achieve a native conformation or to be degraded. In this way, E. coli ensures the presence of sufficient functional protein albeit at a considerable energetic cost.

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. The resulting promoter-engineered isoprenoid genes were combined by serial P1 transductions into a host strain harboring a reporter plasmid containing beta-carotene biosynthesis genes allowing a visual screen for yellow color indicative of beta-carotene accumulation. Construction of an E. coli P(T5)-dxs P(T5)-ispDispF P(T5)-idi P(T5)-ispB strain resulted in producing high titers (6mg/g dry cell weight) of beta-carotene. Surprisingly, over-expression of the ispB gene, which was expected to divert carbon flow from the isoprenoid pathway to quinone biosynthesis, resulted in increased beta-carotene production. We thus demonstrated that chromosomal promoter engineering of the endogenous isoprenoid pathway yielded high levels of beta-carotene in a non-carotenogenic E. coli. The high isoprenoid flux E. coli can be used as a starting strain to produce various carotenoids by introducing heterologous carotenoid genes.

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. coli defenses against superoxide-generating compounds and hydrogen peroxide, respectively, have minor roles. Moreover, additional regulators of the E. coli response to reactive nitrogen species remain to be identified because several of the induced genes were regulated normally in norR, fur, soxRS, and oxyR mutant strains. We propose that the E. coli transcriptional response to reactive nitrogen species is a composite response mediated by the modification of multiple transcription factors containing iron or redox-active cysteines, some specifically designed to sense NO and its derivatives and others that are collaterally activated by the reactive nitrogen species.

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. 4-Azaleucine, a competitive inhibitor of leucyl-tRNA synthetase, surprisingly triggered the heat shock response. Administration of mupirocin, an inhibitor of isoleucyl-tRNA synthetase activity, resulted in changes reminiscent of the stringent response. Treatment with kasugamycin and puromycin (targeting ribosomal subunit association as well as its peptidyl-transferase activity) caused accumulation of mRNAs from ribosomal protein operons. Abundant biosynthetic transcripts were often significantly diminished after treatment with any of these agents. Exposure of a relA strain to mupirocin resulted in accumulation of ribosomal protein operon transcripts. However, the relA strain's response to the other inhibitors was quite similar to that of the wild-type strain.

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. Understanding the transcriptome of an organism includes identifying all transcripts and mapping their 5' and 3' ends. High-density oligonucleotide arrays have enabled the identification of many new transcripts, including small RNAs and antisense RNAs.

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. 3,000) of the 4,228 open reading frames on the microarray remained unchanged; the relative expression of about 320 genes decreased in seawater, whereas that of ca. one fourth (937) increased. Clearly coherent expression patterns were observed for several functional gene groups. Induced genes were numerous in groups specifying the degradation of small molecules (carbon compounds, amino acids and fatty acids), energy metabolism (aerobic and anaerobic respiration, pyruvate dehydrogenase and TCA cycle), chemotaxis and mobility, flagella biosynthesis, surface structures and phage related functions. Repressed genes were clustered in two groups, cell division and nucleotides biosynthesis, indicating a cessation of growth.

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. In addition, UV-B light induced the stringent response in Synechocystis, as indicated by the repression of ribosomal protein transcripts and other mRNAs involved in translation. High-intensity white light- and UV-B-mediated expression profiles overlapped in the down-regulation of photosynthesis genes and induction of heat shock response but differed in several other transcriptional processes including those specifying carbon dioxide uptake and fixation, the stringent response, and the induction profile of the high-light-inducible proteins. These two profile comparisons not only corroborated known physiological changes but also suggested coordinated regulation of many pathways, including synchronized induction of D1 protein recycling and a coupling between decreased phycobilisome biosynthesis and increased phycobilisome degradation. Overall, the gene expression profile analysis generated new insights into the integrated network of genes that adapts rapidly to different wavelengths and intensities of light.

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. To do this, we concentrate upon examples using Escherichia coli and Bacillus subtilis because prior work in these organisms provides a sound basis for comparison.

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. A tolC mutant E. coli host displayed generally lower detection threshold for toxicants. A chromosomal integration of a single copy of the fabA'::lux fusion led to a markedly lower background luminescence, but did not yield an improvement in overall performance. It is proposed that these or similarly constructed reporters of fatty acid biosynthesis inhibition may serve as novel microbial toxicity biosensors.