We applied whole-genome resequencing of Escherichia coli to monitor the acquisition and fixation of mutations that conveyed a selective growth advantage during adaptation to a glycerol-based growth medium. We identified 13 different de novo mutations in five different E. coli strains and monitored their fixation over a 44-d period of adaptation. We obtained proof that the observed spontaneous mutations were responsible for improved fitness by creating single, double and triple site-directed mutants that had growth rates matching those of the evolved strains. The success of this new genome-scale approach indicates that real-time evolution studies will now be practical in a wide variety of contexts.

Genome-scale models of Escherichia coli K-12 MG1655 metabolism have been able to predict growth phenotypes in most, but not all, defined growth environments. Here we introduce the use of an optimization-based algorithm that predicts the missing reactions that are required to reconcile computation and experiment when they disagree. The computer-generated hypotheses for missing reactions were verified experimentally in five cases, leading to the functional assignment of eight ORFs ( yjjLMN, yeaTU, dctA, idnT , and putP ) with two new enzymatic activities and four transport functions. This study thus demonstrates the use of systems analysis to discover metabolic and transport functions and their genetic basis by a combination of experimental and computational approaches.

Our goal is to construct an improved Escherichia coli to serve both as a better model organism and as a more useful technological tool for genome science. We developed techniques for precise genomic surgery and applied them to deleting the largest K-islands of E. coli, identified by comparative genomics as recent horizontal acquisitions to the genome. They are loaded with cryptic prophages, transposons, damaged genes, and genes of unknown function. Our method leaves no scars or markers behind and can be applied sequentially. Twelve K-islands were successfully deleted, resulting in an 8.1% reduced genome size, a 9.3% reduction of gene count, and elimination of 24 of the 44 transposable elements ofE. coli. These are particularly detrimental because they can mutagenize the genome or transpose into clones being propagated for sequencing, as happened in 18 places of the draft human genome sequence. We found no change in the growth rate on minimal medium, confirming the nonessential nature of these islands. This demonstration of feasibility opens the way for constructing a maximally reduced strain, which will provide a clean background for functional genomics studies, a more efficient background for use in biotechnology applications, and a unique tool for studies of genome stability and evolution.[Sequence data described in this paper have been submitted to the DNA Data Bank of Japan, European Molecular Biology Laboratory, and GenBank databases under accession nos. AF402780,AF402779, and AF406953, respectively.]

The genome-wide location of RNA polymerase binding sites was determined inEscherichia coliusing chromatin immunoprecipitation and microarrays (chIP-chip). Cross-linked chromatin was isolated in triplicate from rifampin-treated cells, and DNA bound to RNA polymerase was precipitated with an antibody specific for the β′ subunit. The DNA was amplified and hybridized to “tiled” oligonucleotide microarrays representing the whole genome at 25-bp resolution. A total of 1,139 binding sites were detected and evaluated by comparison to gene expression data from identical conditions and to 961 promoters previously identified by established methods. Of the detected binding sites, 418 were located within 1,000 bp of a known promoter, leaving 721 previously unknown RNA polymerase binding sites. Within 200 bp, we were able to detect 51% (189/368) of the known σ70-specific promoters occurring upstream of an expressed open reading frame and 74% (273/368) within 1,000 bp. Conversely, many known promoters were not detected by chIP-chip, leading to an estimated 26% negative-detection rate. Most of the detected binding sites could be associated with expressed transcription units, but 299 binding sites occurred near inactive transcription units. This map of RNA polymerase binding sites represents a foundation for studies of transcription factors inE. coliand an important evaluation of the chIP-chip technique.