DNA sequence fine-structure analysis of ilvG (IlvG+) mutations of Escherichia coli K-12

Lawther, Robert P.; Calhoun, David H.; Gray, John Edward; Adams, C W; Hauser, Craig A.; Hatfield, G. Wesley

doi:10.1128/jb.149.1.294-298.1982

Cited by 40 publications

(20 citation statements)

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“…In wild type E. coli K-12, ilvG is cryptic. A rho-dependent termination site (15, 26, 52) exists downstream of the site where the ilvG coding sequence is disrupted (14,53). This may result in a major fraction of transcription terminating upstream of ilvE.…”

Section: Discussionmentioning

confidence: 99%

Analysis and comparison of the internal promoter, pE, of the ilvGMEDA operons fromEscherichia coliK-12 andSalmonella typhimurium

Lopes¹,

Lawther²

1986

Nucl Acids Res

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It was previously determined that the distal portion of the ilvGMEDA operon was expressed despite the insertion of transposons into ilvG and ilvE. This observation suggested the existence of internal promoters upstream of ilvE (pE) and ilvD (pD). The internal promoter pE, responsible for part of ilvEDA expression, has been analyzed both in vivo and in vitro. Our results indicate that: pE exists in both E. coli K-12 and S. typhimurium; pE is located in the distal end of the ilvM coding sequence; the pE sequence is highly conserved in the two bacteria; the amino acid sequence of the ilvM gene product is 93% homologous between the two bacteria; transcription from pE can be demonstrated both in vivo and in vitro; the efficiency of pE is essentially equivalent in the two bacteria.

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Section: Discussionmentioning

confidence: 99%

Analysis and comparison of the internal promoter, pE, of the ilvGMEDA operons fromEscherichia coliK-12 andSalmonella typhimurium

Lopes¹,

Lawther²

1986

Nucl Acids Res

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“…Wild-type E. coli K-12 has a naturally occurring frameshift region in the ilvG gene (16,17) that prevents synthesis of the ilvG gene product, a-acetohydroxy acid synthetase, one of three isozymes (12,16,30), and is polar upon the downstream ilvEDA products. Mutant derivatives with 1-base pair deletions or 2-base pair insertions localized within a 10-base pair region of the wild-type ilvG gene (16,17) lead to the synthesis of the synthetase and relief of polarity on ilvEDA. Since this isozyme confers the valine-resistant (Val') growth phenotype, the wildtype cells (ilvG+) are phenotypically IlvG-and Vals, whereas the mutant derivatives (ilvG) are phenotypically IlvG+ and Valr.…”

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confidence: 99%

Absence of significant membrane localization of the proteins coded by the ilvGEDAC genes of Escherichia coli K-12

Gray¹,

Calhoun²

1982

J Bacteriol

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We previously characterized a set of X dilv phages by genetic, restriction enzyme, and heteroduplex analyses and tentatively correlated isoleucine-valine gene products with specific ilv DNA segments by using cloned ilv segments in maxicells and A dilv phage infection of UV-irradiated cells. In this work, the identity of the ilvC gene product, a-acetohydroxy acid isomeroreductase, was confirmed by demonstrating its induction by the physiological inducers aacetolactate and a-acetohydroxybutyrate. The identity of the ilvE gene product, transaminase B, was confirmed by antibody precipitation of the purified enzyme. Phage derivatives with ilv regulatory mutations were found to have the predicted effect upon the ilvGEDA and ilvC protein products. The distribution of the ilvGEDA and ilvC gene products in the soluble, periplasmic, inner membrane, and outer membrane fractions was examined, and no significant membrane association was observed. The expression of the ilv genes in the X dilv phage from ilv and phage X promoters was compared in order to determine the fractional contribution of each to ilv gene expression. An additional protein of 54,000 daltons that was not detected in the previous analysis was observed to be coded by a bacterial gene but was produced only by readthrough from phage promoters.

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“…K12 contains three acetohydroxy acid synthases ( ilvB, ilvG, ilvH ) that are involved in branch-chained amino acid biosynthesis. K-12 does not express ilvG due to a natural frameshift mutation and thus exhibits a growth defect in the presence of exogenous valine and the absence of isoleucine (35, 36). This valine-sensitive growth phenotype is alleviated by restoration of ilvG (37).…”

Section: Resultsmentioning

confidence: 99%

A versatile platform strain for high-fidelity multiplex genome editing

Egbert

Rishi

Adler

et al. 2018

Preprint

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21Precision genome editing accelerates the discovery of the genetic determinants of phenotype and the 22 engineering of novel behaviors in organisms. Advances in DNA synthesis and recombineering have 23 enabled high-throughput engineering of genetic circuits and biosynthetic pathways via directed 24 mutagenesis of bacterial chromosomes. However, the highest recombination efficiencies have to date 25 been reported in persistent mutator strains, which suffer from reduced genomic fidelity. The absence of 26 inducible transcriptional regulators in these strains also prevents concurrent control of genome 27 engineering tools and engineered functions. Here, we introduce a new recombineering platform strain, 28BioDesignER, which incorporates (1) a refactored λ-Red recombination system that reduces toxicity 29 and accelerates multi-cycle recombination, (2) genetic modifications that boost recombination 30 efficiency, and (3) four independent inducible regulators to control engineered functions. These 31 modifications resulted in single-cycle recombineering efficiencies of up to 25% with a seven-fold 32 increase in recombineering fidelity compared to the widely used recombineering strain EcNR2. To 33 facilitate genome engineering in BioDesignER, we have curated eight context-neutral genomic loci,34 termed Safe Sites, for stable gene expression and consistent recombination efficiency. BioDesignER is 35 a platform to develop and optimize engineered cellular functions and can serve as a model to implement 36 comparable recombination and regulatory systems in other bacteria. 37 INTRODUCTION 38The design-build-test (DBT) cycle is a common paradigm used in engineering disciplines. Within the 39 context of synthetic biology it is employed to engineer user-defined cellular functions for applications 40 such as metabolic engineering, biosensing, and therapeutics (1, 2). The rapid prototyping of engineered 41 functions has been facilitated by advances in in vitro DNA assembly, and plasmids have traditionally 42 been used to implement designs in vivo given their ease-of-assembly and portability. However, for 43 deployment in contexts beyond the laboratory such as large-scale industrial bioprocesses or among 44 complex microbial communities, plasmid-based circuits suffer from multiple limitations: high intercellular 45 variation in gene expression, genetic instability from random partitioning of plasmids during cell division, 46 and plasmid loss in environments for which antibiotic use could disrupt native microbial communities or 47 is economically infeasible (3, 4). These shortcomings can be ameliorated once a design is transferred 48 from a plasmid to the host genome, which offers improved genetic stability and lower expression 49 variation (5) along with reduced metabolic load (6). However, behaviors optimized for plasmid contexts 50 often do not map predictably to the genome. As such, building and testing designs directly on the 51 genome can reduce the DBT cycle time and facilitate engineering cellular programs for complex 52 e...

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DNA sequence fine-structure analysis of ilvG (IlvG+) mutations of Escherichia coli K-12

Cited by 40 publications

References 20 publications

Analysis and comparison of the internal promoter, pE, of the ilvGMEDA operons fromEscherichia coliK-12 andSalmonella typhimurium

Analysis and comparison of the internal promoter, pE, of the ilvGMEDA operons fromEscherichia coliK-12 andSalmonella typhimurium

Absence of significant membrane localization of the proteins coded by the ilvGEDAC genes of Escherichia coli K-12

A versatile platform strain for high-fidelity multiplex genome editing

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