Diatoms dominate the biomass of phytoplankton in nutrient-rich conditions and form the basis of some of the world's most productive marine food webs. The diatom nuclear genome contains genes with bacterial and plastid origins as well as genes of the secondary endosymbiotic host (the exosymbiont), yet little is known about the relative contribution of each gene group to diatom metabolism. Here we show that the exosymbiont-derived ornithine-urea cycle, which is similar to that of metazoans but is absent in green algae and plants, facilitates rapid recovery from prolonged nitrogen limitation. RNA-interference-mediated knockdown of a mitochondrial carbamoyl phosphate synthase impairs the response of nitrogen-limited diatoms to nitrogen addition. Metabolomic analyses indicate that intermediates in the ornithine-urea cycle are particularly depleted and that both the tricarboxylic acid cycle and the glutamine synthetase/glutamate synthase cycles are linked directly with the ornithine-urea cycle. Several other depleted metabolites are generated from ornithine-urea cycle intermediates by the products of genes laterally acquired from bacteria. This metabolic coupling of bacterial- and exosymbiont-derived proteins seems to be fundamental to diatom physiology because the compounds affected include the major diatom osmolyte proline and the precursors for long-chain polyamines required for silica precipitation during cell wall formation. So far, the ornithine-urea cycle is only known for its essential role in the removal of fixed nitrogen in metazoans. In diatoms, this cycle serves as a distribution and repackaging hub for inorganic carbon and nitrogen and contributes significantly to the metabolic response of diatoms to episodic nitrogen availability. The diatom ornithine-urea cycle therefore represents a key pathway for anaplerotic carbon fixation into nitrogenous compounds that are essential for diatom growth and for the contribution of diatoms to marine productivity.

EcoCyc (http://EcoCyc.org) provides a comprehensive encyclopedia of Escherichia coli biology. EcoCyc integrates information about the genome, genes and gene products; the metabolic network; and the regulatory network of E. coli. Recent EcoCyc developments include a new initiative to represent and curate all types of E. coli regulatory processes such as attenuation and regulation by small RNAs. EcoCyc has started to curate Gene Ontology (GO) terms for E. coli and has made a dataset of E. coli GO terms available through the GO Web site. The curation and visualization of electron transfer processes has been significantly improved. Other software and Web site enhancements include the addition of tracks to the EcoCyc genome browser, in particular a type of track designed for the display of ChIP-chip datasets, and the development of a comparative genome browser. A new Genome Omics Viewer enables users to paint omics datasets onto the full E. coli genome for analysis. A new advanced query page guides users in interactively constructing complex database queries against EcoCyc. A Macintosh version of EcoCyc is now available. A series of Webinars is available to instruct users in the use of EcoCyc.

Primary productivity of open ocean environments, such as those inhabited by marine picocyanobacteria, is often limited by low inorganic phosphate (P). To observe how these organisms cope with P starvation, we constructed a full genome microarray for Synechococcus sp. WH8102 and compared differences in gene expression under P-replete and P-limited growth conditions, including both early P stress, during extracellular alkaline phosphatase induction, and late P stress. A total of 36 genes showed significant upregulation (4log 2 fold) whereas 23 genes were highly downregulated at the early time point; however, these changes in expression were maintained during late P stress for only 5 of the upregulated genes. Knockout mutants were constructed for genes SYNW0947 and SYNW0948, comprising a two-component regulator hypothesized to have a key function in regulating P metabolism. A high degree of overlap in the sets of genes affected by P stress conditions and in the knockout mutants supports this hypothesis; however, there is some indication that other regulators may be involved in this response in Synechococcus sp. WH8102. Consistent with what has been observed in many other cyanobacteria, the Pho regulon of this strain is comprised largely of genes for alkaline phosphatases, P transport or P metabolism. Interestingly, however, the exact composition and arrangement of the Pho regulon appears highly variable in marine cyanobacteria.

Copper appears to be influencing the distribution and abundance of phytoplankton in marine environments, and cyanobacteria are thought to be the most sensitive of the phytoplankton groups to copper toxicity. By using growth assays of phylogenetically divergent clades, we found that coastal strains of marine Synechococcus species were more tolerant to copper shock than open-ocean strains. The global transcriptional response to two levels of copper shock were determined for both a coastal strain and an open-ocean strain of marine Synechococcus species using whole-genome expression microarrays. Both strains showed an osmoregulatory-like response, perhaps as a result of increasing membrane permeability. This could have implications for marine carbon cycling if copper shock leads to dissolved organic carbon leakage in Synechococcus species. The two strains additionally showed a common reduction in levels of photosynthesis-related gene transcripts. Contrastingly, the open-ocean strain showed a general stress response, whereas the coastal strain exhibited a more specifically oxidative or heavy-metal acclimation response that may be conferring tolerance. In addition, the coastal strain activated more regulatory elements and transporters, many of which are not conserved in other marine Synechococcus strains and may have been acquired by horizontal gene transfer. Thus, tolerance to copper shock in some marine Synechococcus strains may in part be a result of a generally increased ability to sense and respond in a more stress-specific manner.Marine Synechococcus and Prochlorococcus species are together thought to be responsible for at least 20% of global carbon fixation (25). They play such a significant role in the carbon cycle that it is essential to understand to which environmental stresses they are susceptible and how they respond. Cyanobacteria have been shown to be particularly sensitive to copper, for example, compared to other marine phytoplanktons (9). As a group, cyanobacteria have various levels of copper tolerance, with Prochlorococcus species thought to be more sensitive to copper than Synechococcus species (27). Within Synechococcus species, strain tolerance has been shown to vary (9), but those experiments with equilibrated copper levels were done prior to a better understanding of Synechococcus phylogenetic diversity.Applying a copper "shock" seems just as environmentally relevant as using equilibrated copper levels, since there are several mechanisms by which we could see sharp, rapid spikes of Cu 2ϩ concentrations in the surface waters where marine Synechococcus strains are found. Although the Cu 2ϩ concentration is generally low (10 Ϫ14 M) in the surface waters of the open ocean, it can increase by 3 or 4 orders of magnitude below the euphotic zone (12), and a mixing event could thus cause a sharp increase in Cu 2ϩ concentrations. Furthermore, in coastal environments, a sharp spike in free-copper levels can occur after a heavy rain due mostly to anthropogenic inputs (6). Finally, there is evidence that d...

The deluge of data generated by genome sequencing has led to an increasing reliance on bioinformatic predictions, since the traditional experimental approach of characterizing gene function one at a time cannot possibly keep pace with the sequence-based discovery of novel genes. We have utilized Biolog phenotype MicroArrays to identify phenotypes of gene knockout mutants in the opportunistic pathogen and versatile soil bacterium Pseudomonas aeruginosa in a relatively high-throughput fashion. Seventy-eight P. aeruginosa mutants defective in predicted sugar and amino acid membrane transporter genes were screened and clear phenotypes were identified for 27 of these. In all cases, these phenotypes were confirmed by independent growth assays on minimal media. Using qRT-PCR, we demonstrate that the expression levels of 11 of these transporter genes were induced from 4- to 90-fold by their substrates identified via phenotype analysis. Overall, the experimental data showed the bioinformatic predictions to be largely correct in 22 out of 27 cases, and led to the identification of novel transporter genes and a potentially new histamine catabolic pathway. Thus, rapid phenotype identification assays are an invaluable tool for confirming and extending bioinformatic predictions.