Isolation and preliminary characterization of periplasmic-leaky mutants of Escherichia coli K-12

Lazzaroni, J

doi:10.1016/0378-1097(79)90162-9

Cited by 17 publications

(34 citation statements)

References 6 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, these “periplasmic leaky” screens were performed in the pre-genomic era and only identified a small handful of mutants, some of which were never precisely mapped [15]–[18]. We therefore thought that revisiting this genetic approach with our current knowledge and technology would be fruitful for the identification of new factors involved in the biogenesis of the Gram-negative envelope.…”

Section: Introductionmentioning

confidence: 99%

A Genome-Wide Screen for Bacterial Envelope Biogenesis Mutants Identifies a Novel Factor Involved in Cell Wall Precursor Metabolism

et al. 2014

View full text Add to dashboard Cite

The cell envelope of Gram-negative bacteria is a formidable barrier that is difficult for antimicrobial drugs to penetrate. Thus, the list of treatments effective against these organisms is small and with the rise of new resistance mechanisms is shrinking rapidly. New therapies to treat Gram-negative bacterial infections are therefore sorely needed. This goal will be greatly aided by a detailed mechanistic understanding of envelope assembly. Although excellent progress in the identification of essential envelope biogenesis systems has been made in recent years, many aspects of the process remain to be elucidated. We therefore developed a simple, quantitative, and high-throughput assay for mutants with envelope biogenesis defects and used it to screen an ordered single-gene deletion library of Escherichia coli. The screen was robust and correctly identified numerous mutants known to be involved in envelope assembly. Importantly, the screen also implicated 102 genes of unknown function as encoding factors that likely impact envelope biogenesis. As a proof of principle, one of these factors, ElyC (YcbC), was characterized further and shown to play a critical role in the metabolism of the essential lipid carrier used for the biogenesis of cell wall and other bacterial surface polysaccharides. Further analysis of the function of ElyC and other hits identified in our screen is likely to uncover a wealth of new information about the biogenesis of the Gram-negative envelope and the vulnerabilities in the system suitable for drug targeting. Moreover, the screening assay described here should be readily adaptable to other organisms to study the biogenesis of different envelope architectures.

show abstract

Section: Introductionmentioning

confidence: 99%

A Genome-Wide Screen for Bacterial Envelope Biogenesis Mutants Identifies a Novel Factor Involved in Cell Wall Precursor Metabolism

et al. 2014

View full text Add to dashboard Cite

show abstract

“…First approaches mainly focused on characteristics like morphology, ability to release different proteins, as well as sensitivity toward antibiotics and detergents [166][167][168]. First approaches mainly focused on characteristics like morphology, ability to release different proteins, as well as sensitivity toward antibiotics and detergents [166][167][168].…”

Section: Permanently Improved Permeability By Strain Development Mutmentioning

confidence: 99%

Secretion of recombinant proteins from E. coli

Kleiner-Grote

Risse

Friehs

2018

Engineering in Life Sciences

View full text Add to dashboard Cite

The microorganism Escherichia coli is commonly used for recombinant protein production. Despite several advantageous characteristics like fast growth and high protein yields, its inability to easily secrete recombinant proteins into the extracellular medium remains a drawback for industrial production processes. To overcome this limitation, a multitude of approaches to enhance the extracellular yield and the secretion efficiency of recombinant proteins have been developed in recent years. Here, a comprehensive overview of secretion mechanisms for recombinant proteins from E. coli is given and divided into three main sections. First, the structure of the E. coli cell envelope and the known natural secretion systems are described. Second, the use and optimization of different one‐ or two‐step secretion systems for recombinant protein production, as well as further permeabilization methods are discussed. Finally, the often‐overlooked role of cell lysis in secretion studies and its analysis are addressed. So far, effective approaches for increasing the extracellular protein concentration to more than 10 g/L and almost 100% secretion efficiency exist, however, the large range of optimization methods and their combinations suggests that the potential for secretory protein production from E. coli has not yet been fully realized.

show abstract

“…14 The Tol-Pal assembly is coupled to the proton motive force across the IM, 15 and this coupling is required for its primary function of maintaining OM stability, which is most likely expressed at cell division sites to which the assembly is recruited. 16,17 The assembly is essential in Gramnegative bacteria such as Caulobacter crescentus 18 and is required for virulence in pathogenic organisms such as Erwinia chrysanthemi 19 and Salmonella typhimurium. 20,21 Although not essential in E. coli, deletion of tolAQRB genes leads to cell chaining, blebbing of the OM, leakage of periplasmic components to the environment, increased antibiotic and detergent sensitivity, 22 and resistance to colicins and bacteriophages.…”

Section: Introductionmentioning

confidence: 99%

Kinetic Basis for the Competitive Recruitment of TolB by the Intrinsically Disordered Translocation Domain of Colicin E9

Papadakos¹,

Housden²,

Lilly³

et al. 2012

Journal of Molecular Biology

View full text Add to dashboard Cite

TolB and Pal are members of the Tol-Pal system that spans the cell envelope of Gram-negative bacteria and contributes to the stability and integrity of the bacterial outer membrane (OM). Lipoylated Pal is tethered to the OM and binds the β-propeller domain of periplasmic TolB, which, as recent evidence suggests, disengages TolB from its interaction with other components of the Tol system in the inner membrane. Antibacterial nuclease colicins such as colicin E9 (ColE9) also bind the β-propeller domain of TolB in order to catalyze their translocation across the bacterial OM. In contrast to Pal, however, colicin binding to TolB promotes its interaction with other components of the Tol system. Here, through a series of pre-steady-state kinetic experiments utilizing fluorescence resonance energy transfer pairs within the individual protein-protein complexes, we establish the kinetic basis for such 'competitive recruitment' by the TolB-binding epitope (TBE) of ColE9. Surprisingly, the 16-residue disordered ColE9 TBE associates more rapidly with TolB than Pal, a folded 13-kDa protein. Moreover, we demonstrate that calcium ions, which bind within the confines of the TolB β-propeller domain tunnel and are known to increase the affinity of the TolB-ColE9 complex, do not exert their influence through long-range electrostatic effects, as had been predicted, but through short-range effects that slow the dissociation rate of ColE9 TBE from its complex with TolB. Our study demonstrates that an intrinsically disordered protein undergoing binding-induced folding can compete effectively with a globular protein for a common target by associating more rapidly than the globular protein.

show abstract

Isolation and preliminary characterization of periplasmic-leaky mutants of Escherichia coli K-12

Cited by 17 publications

References 6 publications

A Genome-Wide Screen for Bacterial Envelope Biogenesis Mutants Identifies a Novel Factor Involved in Cell Wall Precursor Metabolism

A Genome-Wide Screen for Bacterial Envelope Biogenesis Mutants Identifies a Novel Factor Involved in Cell Wall Precursor Metabolism

Secretion of recombinant proteins from E. coli

Kinetic Basis for the Competitive Recruitment of TolB by the Intrinsically Disordered Translocation Domain of Colicin E9

Contact Info

Product

Resources

About