Comparison of the Local Concentration of the HU Protein for Particular Regions of Genomic DNA in Escherichia coli Cells in Vivo

2005 ◽  
Vol 39 (4) ◽  
pp. 585-592 ◽  
Author(s):  
O. V. Preobrajenskaya ◽  
E. S. Starodubova ◽  
V. L. Karpov ◽  
J. Rouviere-Yaniv
Keyword(s):  
Escherichia Coli ◽  
Genomic Dna ◽  
Local Concentration ◽  
Hu Protein ◽  
Escherichia Coli Cells

An Efficient Site-Directed Mutagenesis Method In Vivo in Escherichia Coli

2012 ◽  
Vol 195-196 ◽  
pp. 407-411
Author(s):  
Mu Qing Qiu
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Recombinant Plasmid ◽  
Genomic Dna ◽  
Gene Replacement ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

In order to develop an efficient site-directed mutagenesis method in vivo, the tests were tested by the following methods. The methods that the fragment knockouted ompR gene was constructed through overlapping PCR, digested by Notand Sal, ligated to plasmid pKOV were applied. The recombination plasmid was transformed into Escherichia coli WMC-001 strain, integrated into the genomic DNA through two step homologous recombination. The Escherichia coli WMC-001/ompR-mutant was obtained due to gene replacement. The fragment of the mutant ompR gene was amplified through overlapping PCR, cloned into pKOV vector. The recombinant plasmid was introduced into Escherichia coli WMC-001/ompR-mutant. The Escherichia coli WMC-001/ompR mutant was also obtained due to gene replacement. Results: The site-directed mutagenesis has been successfully constructed in the ompR gene by sequencing. Conclusion: The method is effective for construction of gene site-directed mutagenesis in vivo.

scholarly journals Bacterial protein translocation requires only one copy of the SecY complex in vivo

2012 ◽  
Vol 198 (5) ◽  
pp. 881-893 ◽  
Author(s):  
Eunyong Park ◽  
Tom A. Rapoport
Keyword(s):  
Escherichia Coli ◽  
Small Molecules ◽  
Protein Translocation ◽  
Cross Linking ◽  
Bacterial Protein ◽  
Oligomeric State ◽  
E Coli ◽  
Cotranslational Translocation ◽  
Escherichia Coli Cells

The transport of proteins across the plasma membrane in bacteria requires a channel formed from the SecY complex, which cooperates with either a translating ribosome in cotranslational translocation or the SecA ATPase in post-translational translocation. Whether translocation requires oligomers of the SecY complex is an important but controversial issue: it determines channel size, how the permeation of small molecules is prevented, and how the channel interacts with the ribosome and SecA. Here, we probe in vivo the oligomeric state of SecY by cross-linking, using defined co- and post-translational translocation intermediates in intact Escherichia coli cells. We show that nontranslocating SecY associated transiently through different interaction surfaces with other SecY molecules inside the membrane. These interactions were significantly reduced when a translocating polypeptide inserted into the SecY channel co- or post-translationally. Mutations that abolish the interaction between SecY molecules still supported viability of E. coli. These results show that a single SecY molecule is sufficient for protein translocation.

scholarly journals Lowering S-Adenosylmethionine Levels inEscherichia coli Modulates C-to-T Transition Mutations

2001 ◽  
Vol 183 (3) ◽  
pp. 921-927 ◽  
Author(s):  
Georgina Macintyre ◽  
C. Victoria Atwood ◽  
Claire G. Cupples
Keyword(s):  
Escherichia Coli ◽  
Dna Methylation ◽  
Genomic Dna ◽  
Synthetase Activity ◽  
Inescherichia Coli ◽  
Genome Methylation ◽  
Sam Synthetase ◽  
Methyl Cytosine

ABSTRACT Deoxycytosine methylase (Dcm) enzyme activity causes mutagenesis in vitro either directly by enzyme-induced deamination of cytosine to uracil in the absence of the methyl donor,S-adenosylmethionine (SAM), or indirectly through spontaneous deamination of [5-methyl]cytosine to thymine. Using a Lac reversion assay, we investigated the contribution of the first mechanism to Dcm mutagenesis in vivo by lowering the levels of SAM.Escherichia coli SAM levels were lowered by reducing SAM synthetase activity via the introduction of a metK84 allele or by hydrolyzing SAM using the bacteriophage T3 SAM hydrolase. ThemetK84 strains exhibited increased C-to-T mutagenesis. Expression of the T3 SAM hydrolase gene, under the control of the arabinose-inducible PBAD promoter, effectively reduced Dcm-mediated genomic DNA methylation. However, increased mutagenesis was not observed until extremely high arabinose concentrations were used, and genome methylation at Dcm sites was negligible.

Bacterial in-cell NMR of human α-synuclein: a disordered monomer by nature?

2012 ◽  
Vol 40 (5) ◽  
pp. 950-954 ◽  
Author(s):  
Andres Binolfi ◽  
Francois-Xavier Theillet ◽  
Philipp Selenko
Keyword(s):  
Escherichia Coli ◽  
Protein Structure ◽  
Intracellular Protein ◽  
E Coli ◽  
Intrinsically Disordered ◽  
Escherichia Coli Cells ◽  
Rule Out ◽  
In Cells

The notion that human α-synuclein is an intrinsically disordered monomeric protein was recently challenged by a postulated α-helical tetramer as the physiologically relevant protein structure. The fact that this alleged conformation had evaded detection for so many years was primarily attributed to a widely used denaturation protocol to purify recombinant α-synuclein. In the present paper, we provide in-cell NMR evidence obtained directly in intact Escherichia coli cells that challenges a tetrameric conformation under native in vivo conditions. Although our data cannot rule out the existence of other intracellular protein states, especially in cells of higher organisms, they indicate clearly that inside E. coli α-synuclein is mostly monomeric and disordered.

scholarly journals Structure of hibernating ribosomes studied by cryoelectron tomography in vitro and in situ

2010 ◽  
Vol 190 (4) ◽  
pp. 613-621 ◽  
Author(s):  
Julio O. Ortiz ◽  
Florian Brandt ◽  
Valério R.F. Matias ◽  
Lau Sennels ◽  
Juri Rappsilber ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Spatial Organization ◽  
Three Dimensional ◽  
E Coli ◽  
Escherichia Coli Cells ◽  
Three Dimensional Configuration

Ribosomes arranged in pairs (100S) have been related with nutritional stress response and are believed to represent a “hibernation state.” Several proteins have been identified that are associated with 100S ribosomes but their spatial organization has hitherto not been characterized. We have used cryoelectron tomography to reveal the three-dimensional configuration of 100S ribosomes isolated from starved Escherichia coli cells and we have described their mode of interaction. In situ studies with intact E. coli cells allowed us to demonstrate that 100S ribosomes do exist in vivo and represent an easily reversible state of quiescence; they readily vanish when the growth medium is replenished.

scholarly journals Simultaneous Binding of Multiple EF-Tu Copies to Translating Ribosomes in Live Escherichia coli

mBio ◽  
2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Mainak Mustafi ◽  
James C. Weisshaar
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Single Molecule ◽  
Ternary Complex ◽  
Elongation Factor ◽  
Ternary Complexes ◽  
Local Concentration ◽  
E Coli ◽  
A Site

ABSTRACT In bacteria, elongation factor Tu is a translational cofactor that forms ternary complexes with aminoacyl-tRNA (aa-tRNA) and GTP. Binding of a ternary complex to one of four flexible L7/L12 units on the ribosome tethers a charged tRNA in close proximity to the ribosomal A site. Two sequential tests for a match between the aa-tRNA anticodon and the current mRNA codon then follow. Because one elongation cycle can occur in as little as 50 ms and the vast majority of aa-tRNA copies are not cognate with the current mRNA codon, this testing must occur rapidly. We present a single-molecule localization and tracking study of fluorescently labeled EF-Tu in live Escherichia coli. Imaging at 2 ms/frame distinguishes 60% slowly diffusing EF-Tu copies (assigned as transiently bound to translating ribosome) from 40% rapidly diffusing copies (assigned as a mixture of free ternary complexes and free EF-Tu). Combining these percentages with copy number estimates, we infer that the four L7/L12 sites are essentially saturated with ternary complexes in vivo. The results corroborate an earlier inference that all four sites can simultaneously tether ternary complexes near the A site, creating a high local concentration that may greatly enhance the rate of testing of aa-tRNAs. Our data and a combinatorial argument both suggest that the initial recognition test for a codon-anticodon match occurs in less than 1 to 2 ms per aa-tRNA copy. The results refute a recent study (A. Plochowietz, I. Farrell, Z. Smilansky, B. S. Cooperman, and A. N. Kapanidis, Nucleic Acids Res 45:926–937, 2016, https://doi.org/10.1093/nar/gkw787) of tRNA diffusion in E. coli that inferred that aa-tRNAs arrive at the ribosomal A site as bare monomers, not as ternary complexes. IMPORTANCE Ribosomes catalyze translation of the mRNA codon sequence into the corresponding sequence of amino acids within the nascent polypeptide chain. Polypeptide elongation can be as fast as 50 ms per added amino acid. Each amino acid arrives at the ribosome as a ternary complex comprising an aminoacyl-tRNA (aa-tRNA), an elongation factor called EF-Tu, and GTP. There are 43 different aa-tRNAs in use, only one of which typically matches the current mRNA codon. Thus, ternary complexes must be tested very rapidly. Here we use fluorescence-based single-molecule methods that locate and track single EF-Tu copies in E. coli. Fast and slow diffusive behavior determines the fraction of EF-Tu copies that are ribosome bound. We infer simultaneous tethering of ~4 ternary complexes to the ribosome, which may facilitate rapid initial testing for codon matching on a time scale of less than 1 to 2 ms per aa-tRNA.

scholarly journals Nucleoids Dynamic in Escherichia coli: A Growth Phase Dependent Process

1970 ◽  
Vol 23 (2) ◽  
pp. 81-88 ◽  
Author(s):  
Ali Azam Talukder ◽  
M Anwar Hossain ◽  
Mamoru Yamada ◽  
Akira Ishihama
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Gene Silencing ◽  
Cell Survival ◽  
Growth Phase ◽  
Genomic Dna ◽  
Bacterial Dna ◽  
Escherichia Coli Cells ◽  
Dna Organization ◽  
Nucleoid Proteins

Bacterial DNA compacts in nucleoid bodies. The organization of nucleoid body depends on the association of genomic DNA with a numbers of histone-like proteins. The relax nucleoids organization in rapidly growing Escherichia coli cells associate with six major proteins, Fis, HU, Hfq, H-NS, StpA and IHF, but at stationary phase the nucleoids further tightly pack with Dps. The final steps of compact nucleoids formation occurs with association of MukBEF complex - a bacterial condensin. The change of nucleoid proteins composition in stationary phase accompanies compact DNA organization and genes silencing. Thus, compact nucleoid organization and gene silencing may be crucial for cell survival in stationary phase.Keywords: Escherichia coli, Nucleoid body, Nucleoid proteins, Nucleoid compaction, CondensinDOI: http://dx.doi.org/10.3329/bjm.v23i2.867 Bangladesh J Microbiol, Volume 23, Number 2, December 2006, pp 81-88

scholarly journals Effect of Different Crowding Agents on the Architectural Properties of the Bacterial Nucleoid-Associated Protein HU

2020 ◽  
Vol 21 (24) ◽  
pp. 9553
Author(s):  
Szu-Ning Lin ◽  
Gijs J.L. Wuite ◽  
Remus T. Dame
Keyword(s):  
Local Concentration ◽  
Complex Interplay ◽  
Binding Properties ◽  
In Vitro System ◽  
Hu Protein ◽  
Tethered Particle Motion ◽  
Bacterial Nucleoid ◽  
Dna Binding Properties

HU is a nucleoid-associated protein expressed in most eubacteria at a high amount of copies (tens of thousands). The protein is believed to bind across the genome to organize and compact the DNA. Most of the studies on HU have been carried out in a simple in vitro system, and to what extent these observations can be extrapolated to a living cell is unclear. In this study, we investigate the DNA binding properties of HU under conditions approximating physiological ones. We report that these properties are influenced by both macromolecular crowding and salt conditions. We use three different crowding agents (blotting grade blocker (BGB), bovine serum albumin (BSA), and polyethylene glycol 8000 (PEG8000)) as well as two different MgCl2 conditions to mimic the intracellular environment. Using tethered particle motion (TPM), we show that the transition between two binding regimes, compaction and extension of the HU protein, is strongly affected by crowding agents. Our observations suggest that magnesium ions enhance the compaction of HU–DNA and suppress filamentation, while BGB and BSA increase the local concentration of the HU protein by more than 4-fold. Moreover, BGB and BSA seem to suppress filament formation. On the other hand, PEG8000 is not a good crowding agent for concentrations above 9% (w/v), because it might interact with DNA, the protein, and/or surfaces. Together, these results reveal a complex interplay between the HU protein and the various crowding agents that should be taken into consideration when using crowding agents to mimic an in vivo system.

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