Biomedical subjects

Christopher D Herring

Publications and source records attributed to Christopher D Herring.

10 recordsLinked to original sources

PubMed2006

Systems approach to refining genome annotation.

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.

Biological Transport↗

PubMed2006

Comparative genome sequencing of Escherichia coli allows observation of bacterial evolution on a laboratory timescale.

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.

Adaptation, Physiological↗

PubMed2005

The global transcriptional regulatory network for metabolism in Escherichia coli exhibits few dominant functional states.

A principal aim of systems biology is to develop in silico models of whole cells or cellular processes that explain and predict observable cellular phenotypes. Here, we use a model of a genome-scale reconstruction of the integrated metabolic and transcriptional regulatory networks for Escherichia coli, composed of 1,010 gene products, to assess the properties of all functional states computed in 15,580 different growth environments. The set of all functional states of the integrated network exhibits a discernable structure that can be visualized in 3-dimensional space, showing that the transcriptional regulatory network governing metabolism in E. coli responds primarily to the available electron acceptor and the presence of glucose as the carbon source. This result is consistent with recently published experimental data. The observation that a complex network composed of 1,010 genes is organized to achieve few dominant modes demonstrates the utility of the systems approach for consolidating large amounts of genome-scale molecular information about a genome and its regulation to elucidate an organism's preferred environments and functional capabilities.

Bacterial Proteins↗

PubMed2005

In silico design and adaptive evolution of Escherichia coli for production of lactic acid.

The development and validation of new methods to help direct rational strain design for metabolite overproduction remains an important problem in metabolic engineering. Here we show that computationally predicted E. coli strain designs, calculated from a genome-scale metabolic model, can lead to successful production strains and that adaptive evolution of the engineered strains can lead to improved production capabilities. Three strain designs for lactate production were implemented yielding a total of 11 evolved production strains that were used to demonstrate the utility of this integrated approach. Strains grown on 2 g/L glucose at 37 degrees C showed lactate titers ranging from 0.87 to 1.75 g/L and secretion rates that were directly coupled to growth rates.

Adaptation, Physiological↗

PubMed2005

Immobilization of Escherichia coli RNA polymerase and location of binding sites by use of chromatin immunoprecipitation and microarrays.

The genome-wide location of RNA polymerase binding sites was determined in Escherichia coli using 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 beta' 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 sigma70-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 in E. coli and an important evaluation of the chIP-chip technique.

Base Sequence↗

PubMed2004

Global transcriptional effects of a suppressor tRNA and the inactivation of the regulator frmR.

Expression of an amber suppressor tRNA should result in read-through of the 326 open reading frames (ORFs) that terminate with amber stop codons in the Escherichia coli genome, including six pseudogenes. Abnormal extension of an ORF might alter the activities of the protein and have effects on cellular physiology, while suppression of a pseudogene could lead to a gain of function. We used oligonucleotide microarrays to determine if any effects were apparent at the level of transcription in glucose minimal medium. Surprisingly, only eight genes had significantly different expression in the presence of the suppressor. Among these were the genes yaiN, adhC, and yaiM, forming a single putative operon whose likely function is the degradation of formaldehyde. Expression of wild-type yaiN was shown to result in repression of the operon, while a suppression-mimicking allele lacking the amber stop codon and extended 7 amino acids did not. The operon was shown to be induced by formaldehyde, and the genes have been renamed frmR, frmA, and frmB, respectively.

Base Sequence↗

PubMed2004

Conditional lethal amber mutations in essential Escherichia coli genes.

The essential genes of microorganisms encode biological functions important for survival and thus tend to be of high scientific interest. Drugs that interfere with essential functions are likely to be interesting candidates for antimicrobials. However, these genes are hard to study genetically because knockout mutations in them are by definition inviable. We recently described a conditional mutation system in Escherichia coli that uses a plasmid to produce an amber suppressor tRNA regulated by the arabinose promoter. This suppressor was used here in the construction of amber mutations in seven essential E. coli genes. Amber stop codons were introduced as "tagalong" mutations in the flanking DNA of a downstream antibiotic resistance marker by lambda red recombination. The drug marker was removed by expression of I-SceI meganuclease, leaving a markerless mutation. We demonstrate the method with the genes frr, gcpE, lpxC, map, murA, ppa, and rpsA. We were unable to isolate an amber mutation in ftsZ. Kinetics of cell death and morphological changes were measured following removal of arabinose. As expected given the wide range of cellular mechanisms represented, different mutants showed widely different death curves. All of the mutations were bactericidal except the mutation in gcpE, which was bacteriostatic. The strain carrying an amber mutation in murA was by far the most sensitive, showing rapid killing in nonpermissive medium. The MurA protein is critical for peptidoglycan synthesis and is the target for the antibiotic fosfomycin. Such experiments may inexpensively provide valuable information for the identification and prioritization of targets for antibiotic development.

Escherichia coli↗

PubMed2003

Gene replacement without selection: regulated suppression of amber mutations in Escherichia coli.

We have developed a method called "gene gorging" to make precise mutations in the Escherichia coli genome at frequencies high enough (1-15%) to allow direct identification of mutants by PCR or other screen rather than by selection. Gene gorging begins by establishing a donor plasmid carrying the desired mutation in the target cell. This plasmid is linearized by in vivo expression of the meganuclease I-SceI, providing a DNA substrate for lambda Red mediated recombination. This results in efficient replacement of the wild type allele on the chromosome with the modified sequence. We demonstrate gene gorging by introducing amber stop codons into the genes xylA, melA, galK, fucI, citA, ybdO, and lacZ. To compliment this approach we developed an arabinose inducible amber suppressor tRNA. Controlled expression mediated by the suppressor was demonstrated for the lacZ and xylA amber mutants.

Anti-Bacterial Agents↗

PubMed2002

A three-megabase yeast artificial chromosome contig spanning the C57BL mouse Igh locus.

The mouse Ig H chain (Igh) complex locus is composed of >100 gene segments encoding the variable, diversity, joining, and constant portions of the Ab H chain protein. To advance the characterization of this locus and to identify all the V(H) genes, we have isolated the entire region from C57BL/6 and C57BL/10 as a yeast artificial chromosome contig. The mouse Igh locus extends approximately three megabases and contains at least 134 V(H) genes classified in 15 partially interspersed families. Two non-Igh pseudogenes (Odc-rs8 and Rpl32-rs14) were localized in the distal part of the locus. This physical yeast artificial chromosome map will provide important structure and guidance for the sequencing of this large, complex, and highly repetitive locus.

Animals↗

PubMed2002

Engineering a reduced Escherichia coli genome.

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 of E. 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.

Chromosome Deletion↗