We have systematically made a set of precisely defined, single-gene deletions of all nonessential genes in Escherichia coli K-12. Open-reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method for inactivation of chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high-throughput studies, two independent mutants were saved for every deleted gene. These mutants-the 'Keio collection'-provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome-wide testing of mutational effects in a common strain background, E. coli K-12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase (http://ecoli.aist-nara.ac.jp/).

MassBank is the first public repository of mass spectra of small chemical compounds for life sciences (<3000 Da). The database contains 605 electron-ionization mass spectrometry (EI-MS), 137 fast atom bombardment MS and 9276 electrospray ionization (ESI)-MS(n) data of 2337 authentic compounds of metabolites, 11 545 EI-MS and 834 other-MS data of 10,286 volatile natural and synthetic compounds, and 3045 ESI-MS(2) data of 679 synthetic drugs contributed by 16 research groups (January 2010). ESI-MS(2) data were analyzed under nonstandardized, independent experimental conditions. MassBank is a distributed database. Each research group provides data from its own MassBank data servers distributed on the Internet. MassBank users can access either all of the MassBank data or a subset of the data by specifying one or more experimental conditions. In a spectral search to retrieve mass spectra similar to a query mass spectrum, the similarity score is calculated by a weighted cosine correlation in which weighting exponents on peak intensity and the mass-to-charge ratio are optimized to the ESI-MS(2) data. MassBank also provides a merged spectrum for each compound prepared by merging the analyzed ESI-MS(2) data on an identical compound under different collision-induced dissociation conditions. Data merging has significantly improved the precision of the identification of a chemical compound by 21-23% at a similarity score of 0.6. Thus, MassBank is useful for the identification of chemical compounds and the publication of experimental data.

Based on the genomic sequence data of Escherichia coli K-12 strain, we have constructed a complete set of cloned individual genes encoding Histidine-tagged proteins with or without GFP fused for functional genomic analysis. Each clone encodes a protein of predicted ORF attached by Histidines and seven spacer amino acids at the N-terminal end, and five spacer amino acids and GFP at the C-terminal end. SfiI restriction sites are generated at both the N- and C-terminal boundaries of ORF upon cloning, which enables easy transfer of ORF to other vector systems by cutting with SfiI. Expression of cloned ORF is under the control of an IPTG-inducible promoter, which is strictly repressed by lacI(q) repressor gene product. The set of cloned ORFs described here should provide unique resources for systematic functional genomic approaches including (i) construction of DNA microarray, (ii) production and purification of proteins, (iii) analysis of protein localization by monitoring GFP fluorescence and (iv) analysis of protein-protein interaction.

Protein-protein interactions play key roles in protein function and the structural organization of a cell. A thorough description of these interactions should facilitate elucidation of cellular activities, targeted-drug design, and whole cell engineering. A large-scale comprehensive pull-down assay was performed using a His-tagged Escherichia coli ORF clone library. Of 4339 bait proteins tested, partners were found for 2667, including 779 of unknown function. Proteins copurifying with hexahistidine-tagged baits on a Ni 2+

Genome-wide analysis of deoxyadenosine methyltransferase-mediated control of gene expression in Escherichia coliconditions. Thus, Dam-controlled genes are involved in adjusting the metabolic and respiratory pathways and bacterial motility to suit particular environmental conditions. The promoters of most of these Damcontrolled genes were also found to contain GATC sequences that overlap with recognition sites for two global regulators, fumarate nitrate reduction (Fnr) and catabolite activator protein (CRP). We propose that Dam-mediated methylation plays an important role in the global regulation of genes, particularly those with Fnr and CRP binding sites. IntroductionThe 4.6 Mbp Escherichia coli genome encodes about 4300 open reading frames (ORFs) (Blattner et al., 1997), the functions of about 50% of which remain unknown. To understand the global gene regulation of the E. coli genome, its gene expression under various conditions has been comprehensively investigated using transcriptome and proteome analytical methods. This includes the transcriptome analyses using DNA microarrays that were performed to study the gene expression that takes place in response to changing environment conditions, during the heat shock response and as a result of gene disruption Tao et al., 1999;Arfin et al., 2000). In addition, the proteome approach using two-dimensional gel electrophoresis has been performed with strains with mutations in nucleoid proteins such as IHF, H-NS or Fis (Nyström, 1995;Laurent-Winter et al., 1997;Choe et al., 1999), as well as to study the response to environmental stimuli (cold shock, heat shock and exogenous pyrophosphate) (Van Bogelen and Neidhardt, 1990;Biville et al., 1996) and to assess the alterations that occur during the change in growth phase (Nyström et al., 1996).Dam (deoxyadenosine methyltransferase) methylates the adenine residue within 5¢-GATC-3¢ sequences in double-stranded DNA. This methylation is known to play important physiological roles in E. coli. This is particularly demonstrated by Dam-defective mutants, which have highly pleiotropic changes including increased mutability, hyper-recombination and transcriptional alterations (Marinus, 1996;2000). Dam also contributes to the timing at which chromosome replication is initiated (Marinus, 1996). These processes are all regulated by the SummaryDeoxyadenosine methyltransferase (Dam) methylates the deoxyadenine residues in 5¢-GATC-3¢ sequences and is important in many cellular processes in Escherichia coli. We performed a computational analysis of the entire E. coli genome and confirmed that GATC sequences are distributed unevenly in regulatory regions, which suggests that Dam might regulate gene transcription. To test this, a highdensity DNA microarray of 4097 E. coli genes was constructed and used to assess the gene expression profiles of the wild type and the dam-16::kam mutant strain grown under four different conditions. We also used two-dimensional electrophoretic analysis of the proteome to assess the protein profiles. The expressi...