A whole genome approach to in vivo DNA-protein interactions in E. coli

Nature ◽  
1992 ◽  
Vol 360 (6404) ◽  
pp. 606-610 ◽  
Author(s):  
Ming X. Wang ◽  
George M. Church
Keyword(s):  
Protein Interactions ◽  
Whole Genome ◽  
E Coli ◽  
Dna Protein Interactions

scholarly journals In vivo analysis of DNA-protein interactions on the human erythropoietin enhancer.

1997 ◽  
Vol 17 (2) ◽  
pp. 851-856 ◽  
Author(s):  
B Hu ◽  
E Wright ◽  
L Campbell ◽  
K L Blanchard
Keyword(s):  
Oxygen Tension ◽  
Protein Interactions ◽  
Proximal Promoter ◽  
Low Oxygen ◽  
Initiation Rate ◽  
Cobalt Exposure ◽  
In Cells ◽  
Dna Protein Interactions

The erythropoietin (EPO) gene is one of the best examples of a mammalian gene controlled by oxygen tension. The DNA elements responsible for hypoxia-induced transcription consist of a short region of the proximal promoter and a <50-bp 3' enhancer. The elements act cooperatively to increase the transcriptional initiation rate approximately 100-fold in response to low oxygen tension in Hep3B cells. Two distinct types of transactivating proteins have been demonstrated to bind the response elements in the human EPO enhancer in vitro: one shows hypoxia-inducible DNA binding activity, while the other activity binds DNA under normoxic and hypoxic conditions. We have investigated the DNA-protein interactions on the human EPO enhancer in living tissue culture cells that produce EPO in a regulated fashion (Hep3B) and in cells that do not express EPO under any conditions tested (HeLa). We have identified in vivo DNA-protein interactions on the control elements in the human EPO enhancer by ligation-mediated PCR technology. We show that the putative protein binding sites in the EPO enhancer are occupied in vivo under conditions of normoxia, hypoxia, and cobalt exposure in EPO-producing cells. These sites are not occupied in cells that do not produce EPO. We also provide evidence for a conformational change in the topography of the EPO enhancer in response to hypoxia and cobalt exposure.

scholarly journals Evidence for Multimerization of Neu Proteins Involved in Polysialic Acid Synthesis in Escherichia coliK1 Using Improved LexA-Based Vectors

2000 ◽  
Vol 182 (18) ◽  
pp. 5267-5270 ◽  
Author(s):  
Dayle A. Daines ◽  
Richard P. Silver
Keyword(s):  
Protein Interactions ◽  
Polysialic Acid ◽  
Genetic System ◽  
Acid Synthesis ◽  
Full Length ◽  
Protein Protein Interactions ◽  
Protein Fragment ◽  
Reading Frame ◽  
E Coli

ABSTRACT Recently, M. Dmitrova et al. (Mol. Gen. Genet. 257:205–212, 1998) described a LexA-based genetic system to monitor protein-protein interactions in an Escherichia coli background. However, the plasmids used in this system, pMS604 and pDP804, were not readily amenable for general use. In this report, we describe modifications of both plasmids that allow fragments of DNA to be fused to either vector in any reading frame. Homodimerization and heterodimerization of full-length proteins involved in polysialic acid synthesis in E. coli K1, as well as heterodimerization between a full-length protein and a protein fragment, demonstrate the usefulness of the modified plasmids for investigating bacterial protein-protein interactions in vivo.

scholarly journals Whole genome sequencing identifiesin vivoacquisition of ablaCTX-M-27-encoding IncFII transmissible plasmid as the cause of ceftriaxone treatment failure for an invasiveSalmonella entericaserovar Typhimurium infection.

2016 ◽  
pp. AAC.01649-16 ◽  
Author(s):  
Bruce McCollister ◽  
Cassandra V. Kotter ◽  
Daniel N. Frank ◽  
Taylor Washburn ◽  
Michael G. Jobling
Keyword(s):  
Treatment Failure ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Resistant Isolate ◽  
Virulence Plasmid ◽  
Whole Genome ◽  
Transfer Resistance ◽  
E Coli ◽  
Transmissible Plasmid

We report a case of ceftriaxone (CTX) treatment failure for bacteremia caused bySalmonella entericasubsp.entericaserovar Typhimurium, due to thein vivoacquisition of ablaCTX-M-27-encoding IncFII group transmissible plasmid. The original β-lactamase-susceptible isolate ST882S was replaced by the resistant isolate ST931R during ceftriaxone treatment. After relapse, treatment was changed to ciprofloxacin and the patient recovered. Isolate ST931R could transfer resistance toE. coliat 37 °C. We used whole-genome sequencing of ST882S and ST931R, theE. colitransconjugant, and isolated plasmid DNA to unequivocally show that ST882S and ST931R had identical chromosomes, both having identical 206 single nucleotide polymorphisms (SNPs) versusS.Typhimurium 14028s. We assembled a complete circular genome for ST931R, to which ST882S reads mapped with no SNPs. ST882S and ST931R were isogenic except for the presence of 3 additional plasmids in ST931R. ST931R and theE. colitransconjugant were ceftriaxone-resistant due to the presence of a 60.5 kb IS26-flanked-blaCTX-M-27encoding IncFII plasmid. Compared to 14082s, ST931R has almost identical Gifsy-1, Gifsy-2, and ST64B prophages, lacks Gifsy-3 and instead carries a unique Fels-2 prophage related to that found in LT2. ST882S and ST931R both had a 94 kb virulence plasmid showing >99% identity with pSLT14028s, and a cryptic 3904 bp replicon; ST931R also has cryptic 93 kb IncI1 and 62 kb IncI2 group plasmids. To the best of our knowledge,in vivoacquisition of extended spectrum β-lactamase-resistance byS. Typhimurium andblaCTX-M-27genes in US isolates ofSalmonellahave not previously been reported.

scholarly journals Global query on bacterial sirtuin CobB interactants reveals crosstalk with PRPP synthase Prs

2020 ◽  
Author(s):  
Beata M. Walter ◽  
Joanna Morcinek-Orłowska ◽  
Aneta Szulc ◽  
Andrew L. Lovering ◽  
Manuel Banzhaf ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Global Analysis ◽  
Stable Complex ◽  
Protein Protein Interactions ◽  
Cellular Functions ◽  
Nad Metabolism ◽  
E Coli ◽  
Nad Synthesis ◽  
Wide Range

AbstractProtein lysine acetylation regulates a wide range of cellular functions. It is controlled by a family of NAD-dependent protein deacetylases called sirtuins. In eukaryotes, sirtuins activity is coupled to spatiotemporally-controlled NAD+ level, whereas the mechanism of their regulation in bacteria is less clear. E. coli possesses a single sirtuin – CobB. However, it is unclear how CobB activity is coupled to NAD+ metabolism. In this work we show that this coordination is achieved in E. coli cells through a CobB interaction with PRPP synthase Prs, an enzyme necessary for NAD+ synthesis. Employing global analysis of protein-protein interactions formed by CobB, we demonstrate that it forms a stable complex with Prs. This assembly stimulates CobB deacetylase activity and partially protects it from inhibition by nicotinamide. We provide evidence that Prs acetylation is not necessary for CobB binding but affects the global acetylome in vivo. Our results show that CobB ameliorates Prs activity under conditions of Prs cofactors deficiency. Therefore, we propose that CobB-Prs crosstalk orchestrates the NAD+ metabolism and protein acetylation in response to environmental cues.

scholarly journals Transient protein-protein interactions perturb E.coli metabolome and cause gene dosage toxicity

2016 ◽  
Author(s):  
Sanchari Bhattacharyya ◽  
Shimon Bershtein ◽  
Jin Yan ◽  
Tijda Argun ◽  
Amy I. Gilson ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Gene Dosage ◽  
Bacterial Species ◽  
Dynamic Network ◽  
Catalytic Efficiency ◽  
Evolutionary Divergence ◽  
Protein Protein Interactions ◽  
E Coli ◽  
Metabolic Imbalance

Several genes exhibit gene dosage toxicity yet its molecular underpinnings remain unknown. Here we demonstrate that overexpression of DHFR in E. coli causes toxic metabolic imbalance triggered by interactions with several enzymes involved in 1-carbon metabolism, in particular GlyA and PurH. DHFR overexpression partially inhibits activity of these enzymes, but at physiological concentrations, PurH-DHFR interaction enhances catalytic efficiency of DHFR, implying a functional interaction in vivo. Surprisingly, overexpression of orthologous DHFRs from other bacterial species caused minimal metabolic and fitness perturbations, despite pulling out more interacting partners than overexpressed endogenous DHFR. Orthologous DHFRs were less potent in inhibiting E. coli GlyA and PurH, or gaining a catalytic improvement upon interaction with PurH, indicating a partial loss of interaction specificity due to evolutionary divergence. This study shows how protein overexpression perturbs a dynamic network of weak yet potentially functional PPI with consequences for the metabolic state of cells and their fitness.

scholarly journals MCR-1 and OXA-48 In Vivo Acquisition in KPC-Producing Escherichia coli after Colistin Treatment

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Racha Beyrouthy ◽  
Frederic Robin ◽  
Aude Lessene ◽  
Igor Lacombat ◽  
Laurent Dortet ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Content Type ◽  
First Report ◽  
E Coli ◽  
Carbapenem Resistant

ABSTRACT The spread of mcr-1-encoding plasmids into carbapenem-resistant Enterobacteriaceae raises concerns about the emergence of untreatable bacteria. We report the acquisition of mcr-1 in a carbapenem-resistant Escherichia coli strain after a 3-week course of colistin in a patient repatriated to France from Portugal. Whole-genome sequencing revealed that the Klebsiella pneumoniae carbapenemase-producing E. coli strain acquired two plasmids, an IncL OXA-48-encoding plasmid and an IncX4 mcr-1-encoding plasmid. This is the first report of mcr-1 in carbapenemase-encoding bacteria in France.

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