scholarly journals The A-site Finger in 23 S rRNA Acts as a Functional Attenuator for Translocation

2006 ◽  
Vol 281 (43) ◽  
pp. 32303-32309 ◽  
Author(s):  
Taeko Komoda ◽  
Neuza S. Sato ◽  
Steven S. Phelps ◽  
Naoki Namba ◽  
Simpson Joseph ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Functional Role ◽  
Elongation Factor ◽  
Normal Growth ◽  
Wild Type ◽  
Reading Frame ◽  
E Coli ◽  
Translational Activity ◽  
A Site

Helix 38 (H38) in 23 S rRNA, which is known as the “A-site finger (ASF),” is located in the intersubunit space of the ribosomal 50 S subunit and, together with protein S13 in the 30 S subunit, it forms bridge B1a. It is known that throughout the decoding process, ASF interacts directly with the A-site tRNA. Bridge B1a becomes disrupted by the ratchet-like rotation of the 30 S subunit relative to the 50 S subunit. This occurs in association with elongation factor G (EF-G)-catalyzed translocation. To further characterize the functional role(s) of ASF, variants of Escherichia coli ribosomes with a shortened ASF were constructed. The E. coli strain bearing such ASF-shortened ribosomes had a normal growth rate but enhanced +1 frameshift activity. ASF-shortened ribosomes showed normal subunit association but higher activity in poly(U)-dependent polyphenylalanine synthesis than the wild type (WT) ribosome at limited EF-G concentrations. In contrast, other ribosome variants with shortened bridge-forming helices 34 and 68 showed weak subunit association and less efficient translational activity than the WT ribosome. Thus, the higher translational activity of ASF-shortened ribosomes is caused by the disruption of bridge B1a and is not due to weakened subunit association. Single round translocation analyses clearly demonstrated that the ASF-shortened ribosomes have higher translocation activity than the WT ribosome. These observations indicate that the intrinsic translocation activity of ribosomes is greater than that usually observed in the WT ribosome and that ASF is a functional attenuator for translocation that serves to maintain the reading frame.

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 Characterization of an Escherichia coli O157:H7 O-Antigen Deletion Mutant and Effect of the Deletion on Bacterial Persistence in the Mouse Intestine and Colonization at the Bovine Terminal Rectal Mucosa

2008 ◽  
Vol 74 (16) ◽  
pp. 5015-5022 ◽  
Author(s):  
Haiqing Sheng ◽  
Ji Youn Lim ◽  
Maryann K. Watkins ◽  
Scott A. Minnich ◽  
Carolyn J. Hovde
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Cell Membrane ◽  
Membrane Permeability ◽  
Sodium Dodecyl ◽  
Rectal Mucosa ◽  
Cell Membrane Permeability ◽  
Wild Type ◽  
E Coli ◽  
O Antigen

ABSTRACT Escherichia coli O157:H7 causes hemorrhagic colitis and the life-threatening hemolytic-uremic syndrome in humans and transiently colonizes healthy cattle at the terminal rectal mucosa. To investigate the role of the O antigen in persistence and colonization in the animal host, we generated an E. coli O157:H7 mutant defective in the synthesis of the lipopolysaccharide side chain (O antigen) by deletion of a putative perosamine synthetase gene (per) in the rfb cluster. The lack of O antigen was confirmed by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and anti-O157 antibody. The growth rate and cell membrane permeability of the Δper mutant were similar to the growth rate and cell membrane permeability of the wild type. Changes in membrane and secreted proteins were observed, but the expression of intimin, EspA, and EspB, implicated in bacterial intestinal colonization, was not altered, as determined by immunoblotting and reverse transcription-PCR. Similar to other O-antigen deletion mutants, the Δper mutant was pleiotropic for autoaggregation and motility (it was FliC negative as determined by immunoblotting and flagellum negative as determined by electron microscopy). The abilities of the mutant and the wild type to persist in the murine intestine and to colonize the bovine terminal rectal mucosa were compared. Mice fed the Δper mutant shed lower numbers of bacteria (P < 0.05) over a shorter time than mice fed the wild-type or complemented strain. After rectal application in steers, lower numbers of the Δper mutant than of the wild type colonized the rectoanal junction mucosa, and the duration of the colonization was shorter (P < 0.05). Our previous work showed that flagella do not influence E. coli O157:H7 colonization at the bovine terminal rectal mucosa, so the current findings suggest that the O antigen contributes to efficient bovine colonization.

scholarly journals Protein Synthesis in Escherichia coli with Mischarged tRNA

2003 ◽  
Vol 185 (12) ◽  
pp. 3524-3526 ◽  
Author(s):  
Bokkee Min ◽  
Makoto Kitabatake ◽  
Carla Polycarpo ◽  
Joanne Pelaschier ◽  
Gregory Raczniak ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Elongation Factor ◽  
Trna Synthetase ◽  
Wild Type ◽  
Wild Type Level ◽  
E Coli ◽  
Missense Mutant ◽  
Tryptophan Synthetase

ABSTRACT Two types of aspartyl-tRNA synthetase exist: the discriminating enzyme (D-AspRS) forms only Asp-tRNAAsp, while the nondiscriminating one (ND-AspRS) also synthesizes Asp-tRNAAsn, a required intermediate in protein synthesis in many organisms (but not in Escherichia coli). On the basis of the E. coli trpA34 missense mutant transformed with heterologous ND-aspS genes, we developed a system with which to measure the in vivo formation of Asp-tRNAAsn and its acceptance by elongation factor EF-Tu. While large amounts of Asp-tRNAAsn are detrimental to E. coli, smaller amounts support protein synthesis and allow the formation of up to 38% of the wild-type level of missense-suppressed tryptophan synthetase.

scholarly journals Escherichia coli robustly expresses ATP synthase at growth rate maximizing concentrations

2021 ◽  
Author(s):  
Iraes Rabbers ◽  
Frank J Bruggeman
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Protein Expression ◽  
Genetic Variants ◽  
Evolutionary Adaptation ◽  
Wild Type ◽  
Short Term ◽  
E Coli ◽  
Fitness Maximization

AbstractImproved protein expression is an important evolutionary adaptation of bacteria. A key question is whether evolution has led to optimal protein expression that maximizes immediate growth rate (short-term fitness) across conditions. Alternatively, fitter genetic variants could display suboptimal short-term fitness, because they cannot do better or because they strive for long-term fitness maximization by, for instance, anticipating future conditions. To answer this question, we focus on the ATP-producing enzyme F1F0 H+-ATPase, which is an abundant enzyme and ubiquitously expressed across conditions. We tested the optimality of H+-ATPase expression in Escherichia coli across 27 different nutrient conditions. In all tested conditions, wild-type E. coli expresses its H+- ATPase remarkably close to optimal concentrations that maximize immediate growth rate. This work indicates that bacteria can achieve robust optimal protein expression for immediate growth- rate.

scholarly journals Determining the Optimal Thymidine Concentration for Growing Thy−Escherichia coli Strains

1998 ◽  
Vol 180 (11) ◽  
pp. 2992-2994 ◽  
Author(s):  
Felipe Molina ◽  
Alfonso Jiménez-Sánchez ◽  
Elena C. Guzmán
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Growth Medium ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Strain Mg1655 ◽  
E Coli ◽  
Replication Time ◽  
Detectable Difference

ABSTRACT Changes of thymidine concentration in the growth medium affect the chromosome replication time of Thy− strains without at the same time causing a detectable difference in the growth rate (R. H. Pritchard and A. Zaritsky, Nature 226:126–131, 1970). Consequently, the optimal thymidine concentration cannot be determined by ascertaining which concentration produces the highest growth rate. Here we present a method for determining the optimal thymidine concentration of any Thy− Escherichia coli strain. Using this method, we found that the E. coli “wild-type” strain MG1655 has a partial Thy− phenotype.

scholarly journals degS Is Necessary for Virulence and Is among Extraintestinal Escherichia coli Genes Induced in Murine Peritonitis

2003 ◽  
Vol 71 (6) ◽  
pp. 3088-3096 ◽  
Author(s):  
Peter Redford ◽  
Paula L. Roesch ◽  
Rodney A. Welch
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Luria Bertani ◽  
Broth Culture ◽  
Wild Type ◽  
E Coli ◽  
Virulence Attenuation

ABSTRACT Extraintestinal Escherichia coli strains cause meningitis, sepsis, urinary tract infection, and other infections outside the bowel. We examined here extraintestinal E. coli strain CFT073 by differential fluorescence induction. Pools of CFT073 clones carrying a CFT073 genomic fragment library in a promoterless gfp vector were inoculated intraperitoneally into mice; bacteria were recovered by lavage 6 h later and then subjected to fluorescence-activated cell sorting. Eleven promoters were found to be active in the mouse but not in Luria-Bertani (LB) broth culture. Three are linked to genes for enterobactin, aerobactin, and yersiniabactin. Three others are linked to the metabolic genes metA, gltB, and sucA, and another was linked to iha, a possible adhesin. Three lie before open reading frames of unknown function. One promoter is associated with degS, an inner membrane protease. Mutants of the in vivo-induced loci were tested in competition with the wild type in mouse peritonitis. Of the mutants tested, only CFT073 degS was found to be attenuated in peritoneal and in urinary tract infection, with virulence restored by complementation. CFT073 degS shows growth similar to that of the wild type at 37°C but is impaired at 43°C or in 3% ethanol LB broth at 37°C. Compared to the wild type, the mutant shows similar serum survival, motility, hemolysis, erythrocyte agglutination, and tolerance to oxidative stress. It also has the same lipopolysaccharide appearance on a silver-stained gel. The basis for the virulence attenuation is unclear, but because DegS is needed for σE activity, our findings implicate σE and its regulon in E. coli extraintestinal pathogenesis.

scholarly journals Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

2005 ◽  
Vol 71 (7) ◽  
pp. 3468-3474 ◽  
Author(s):  
Gyeong Tae Eom ◽  
Jae Kwang Song ◽  
Jung Hoon Ahn ◽  
Yeon Soo Seo ◽  
Joon Shick Rhee
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Abc Transporter ◽  
Microtiter Plate ◽  
Single Amino Acid ◽  
Wild Type ◽  
E Coli ◽  
Single Amino Acid Change ◽  
Secretion Efficiency

ABSTRACT The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.

scholarly journals Quorum Sensing Is a Global Regulatory Mechanism in Enterohemorrhagic Escherichia coli O157:H7

2001 ◽  
Vol 183 (17) ◽  
pp. 5187-5197 ◽  
Author(s):  
Vanessa Sperandio ◽  
Alfredo G. Torres ◽  
Jorge A. Girón ◽  
James B. Kaper
Keyword(s):  
Escherichia Coli ◽  
Quorum Sensing ◽  
Type Strain ◽  
Wild Type Strain ◽  
Regulatory Mechanism ◽  
Sos Response ◽  
Enterohemorrhagic Escherichia Coli ◽  
Trna Genes ◽  
Wild Type ◽  
E Coli

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is responsible for outbreaks of bloody diarrhea and hemolytic-uremic syndrome in many countries. EHEC virulence mechanisms include the production of Shiga toxins (Stx) and formation of attaching and effacing (AE) lesions on intestinal epithelial cells. We recently reported that genes involved in the formation of the AE lesion were regulated by quorum sensing through autoinducer-2, which is synthesized by the product of the luxS gene. In this study we hybridized an E. coli gene array with cDNA synthesized from RNA that was extracted from EHEC strain 86-24 and its isogenicluxS mutant. We observed that 404 genes were regulated by luxS at least fivefold, which comprises approximately 10% of the array genes; 235 of these genes were up-regulated and 169 were down-regulated in the wild-type strain compared to in theluxS mutant. Down-regulated genes included several involved in cell division, as well as ribosomal and tRNA genes. Consistent with this pattern of gene expression, theluxS mutant grows faster than the wild-type strain (generation times of 37.5 and 60 min, respectively, in Dulbecco modified Eagle medium). Up-regulated genes included several involved in the expression and assembly of flagella, motility, and chemotaxis. Using operon::lacZ fusions to class I, II, and III flagellar genes, we were able to confirm this transcriptional regulation. We also observed fewer flagella by Western blotting and electron microscopy and decreased motility halos in semisolid agar in the luxS mutant. The average swimming speeds for the wild-type strain and the luxS mutant are 12.5 and 6.6 μm/s, respectively. We also observed an increase in the production of Stx due to quorum sensing. Genes encoding Stx, which are transcribed along with λ-like phage genes, are induced by an SOS response, and genes involved in the SOS response were also regulated by quorum sensing. These results indicate that quorum sensing is a global regulatory mechanism for basic physiological functions of E. coli as well as for virulence factors.

scholarly journals exrB: amalB-linked gene inEscherichia coliB involved in sensitivity to radiation and filament formation

1974 ◽  
Vol 23 (2) ◽  
pp. 175-184 ◽  
Author(s):  
Joseph Greenberg ◽  
Leonard J. Berends ◽  
John Donch ◽  
Michael H. L. Green
Keyword(s):  
Escherichia Coli ◽  
Normal Growth ◽  
Filament Formation ◽  
Wild Type ◽  
Sensitive Mutant ◽  
Γ Radiation ◽  
Linked Gene ◽  
Derivatives Of

SUMMARYPAM 26, a radiation-sensitive mutant ofEscherichia colistrain B, is described. Its properties are attributable to a mutation in a gene,exrB, which is cotransducible withmalB. It differs fromuvrA(alsomalB-linked) derivatives of strain B in being sensitive to 1-methyl-3-nitro-1-nitroso-guanidine and γ-radiation, and in being able to reactivate UV-irradiated phage T3. It differs fromexrA(alsomalB-linked) derivatives of strain B in forming filaments during the course of normal growth as well as after irradiation. WhenexrBwas transduced into a K12 (lon+) strain, filaments did not form spontaneously. Three-point transductions established the order of markers asmet A malB exrB. Based on an analysis of the frequency of wild-type recombinants in a reciprocal transduction betweenexrAandexrBstrains, it was inferred that they are not isogenic and that the order of markers ismalB exrA exrB.

O6-methylguanine-DNA methyltransferase in wild-type and ada mutants of Escherichia coli

1982 ◽  
Vol 152 (1) ◽  
pp. 534-537
Author(s):  
S Mitra ◽  
B C Pal ◽  
R S Foote
Keyword(s):  
Escherichia Coli ◽  
Dna Methyltransferase ◽  
Wild Type ◽  
E Coli ◽  
Methylguanine Dna Methyltransferase ◽  
Synthetic Dna ◽  
In The Wild ◽  
Low Levels ◽  
Enzyme Molecules ◽  
Dna Substrate

O(6)-Methylguanine-DNA methyltransferase is induced in Escherichia coli during growth in low levels of N-methyl-N'-nitro-N-nitrosoguanidine. We have developed a sensitive assay for quantitating low levels of this activity with a synthetic DNA substrate containing 3H-labeled O(6)-methylguanine as the only modified base. Although both wild-type and adaptation-deficient (ada) mutants of E. coli contained low but comparable numbers (from 13 to 60) of the enzyme molecules per cell, adaptation treatment caused a significant increase of the enzyme in the wild type but not in the ada mutants, suggesting that the ada mutation is in a regulatory locus and not in the structural gene for the methyltransferase.

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