scholarly journals Contributions of the N- and C-Terminal Domains of Initiation Factor 3 to Its Functions in the Fidelity of Initiation and Antiassociation of the Ribosomal Subunits

2017 ◽  
Vol 199 (11) ◽  
Author(s):  
Shreya Ahana Ayyub ◽  
Divya Dobriyal ◽  
Umesh Varshney
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Bacterial Growth ◽  
Translation Initiation ◽  
Ribosomal Subunit ◽  
Initiation Factor ◽  
Start Codon ◽  
Content Type ◽  
E Coli

ABSTRACT Initiation factor 3 (IF3) is one of the three conserved prokaryotic translation initiation factors essential for protein synthesis and cellular survival. Bacterial IF3 is composed of a conserved architecture of globular N- and C-terminal domains (NTD and CTD) joined by a linker region. IF3 is a ribosome antiassociation factor which also modulates selection of start codon and initiator tRNA. All the functions of IF3 have been attributed to its CTD by in vitro studies. However, the in vivo relevance of these findings has not been investigated. By generating complete and partial IF3 (infC) knockouts in Escherichia coli and by complementation analyses using various deletion constructs, we show that while the CTD is essential for E. coli survival, the NTD is not. Polysome profiles reaffirm that CTD alone can bind to the 30S ribosomal subunit and carry out the ribosome antiassociation function. Importantly, in the absence of the NTD, bacterial growth is compromised, indicating a role for the NTD in the fitness of cellular growth. Using reporter assays for in vivo initiation, we show that the NTD plays a crucial role in the fidelity function of IF3 by avoiding (i) initiation from non-AUG codons and (ii) initiation by initiator tRNAs lacking the three highly conserved consecutive GC pairs (in the anticodon stem) known to function in concert with IF3. IMPORTANCE Initiation factor 3 regulates the fidelity of eubacterial translation initiation by ensuring the formation of an initiation complex with an mRNA bearing a canonical start codon and with an initiator tRNA at the ribosomal P site. Additionally, IF3 prevents premature association of the 50S ribosomal subunit with the 30S preinitiation complex. The significance of our work in Escherichia coli is in demonstrating that while the C-terminal domain alone sustains E. coli for its growth, the N-terminal domain adds to the fidelity of initiation of protein synthesis and to the fitness of the bacterial growth.

scholarly journals Restrictive Streptomycin Resistance Mutations Decrease the Formation of Attaching and Effacing Lesions in Escherichia coli O157:H7 Strains

2013 ◽  
Vol 57 (9) ◽  
pp. 4260-4266 ◽  
Author(s):  
Chun Chen ◽  
Carla A. Blumentritt ◽  
Meredith M. Curtis ◽  
Vanessa Sperandio ◽  
Alfredo G. Torres ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Subunit ◽  
Streptomycin Resistance ◽  
Resistance Mutations ◽  
Content Type ◽  
E Coli ◽  
Enterocyte Effacement ◽  
Translocated Proteins

ABSTRACTStreptomycin binds to the bacterial ribosome and disrupts protein synthesis by promoting misreading of mRNA. Restrictive mutations on the ribosomal subunit protein S12 confer a streptomycin resistance (Strr) phenotype and concomitantly increase the accuracy of the decoding process and decrease the rate of translation. Spontaneous Strrmutants ofEscherichia coliO157:H7 have been generated forin vivostudies to promote colonization and to provide a selective marker for this pathogen. The locus of enterocyte effacement (LEE) ofE. coliO157:H7 encodes a type III secretion system (T3SS), which is required for attaching and effacing to the intestinal epithelium. In this study, we observed decreases in both the expression and secretion levels of the T3SS translocated proteins EspA and EspB inE. coliO157:H7 Strrrestrictive mutants, which have K42T or K42I mutations in S12. However, mildly restrictive (K87R) and nonrestrictive (K42R) mutants showed slight or indistinguishable changes in EspA and EspB secretion. Adherence and actin staining assays indicated that restrictive mutations compromised the formation of attaching and effacing lesions inE. coliO157:H7. Therefore, we suggest thatE. coliO157:H7 strains selected for Strrshould be thoroughly characterized beforein vivoandin vitroexperiments that assay for LEE-directed phenotypes and that strains carrying nonrestrictive mutations such as K42R make better surrogates of wild-type strains than those carrying restrictive mutations.

scholarly journals Measurements of translation initiation from all 64 codons inE. coli

2016 ◽  
Author(s):  
Ariel Hecht ◽  
Jeff Glasgow ◽  
Paul R. Jaschke ◽  
Lukmaan Bawazer ◽  
Matthew S. Munson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Molecular Biology ◽  
Translation Initiation ◽  
Biological Systems ◽  
Start Codon ◽  
Living Matter ◽  
E Coli ◽  
Natural And Synthetic

ABSTRACTOur understanding of translation is one cornerstone of molecular biology that underpins our capacity to engineer living matter. The canonical start codon (AUG) and a few near-cognates (GUG, UUG) are typically considered as the “start codons” for translation initiation inEscherichia coli(E. coli). Translation is typically not thought to initiate from the 61 remaining codons. Here, we systematically quantified translation initiation inE. colifrom all 64 triplet codons. We detected protein synthesis above background initiating from at least 46 codons. Translation initiated from these non-canonical start codons at levels ranging from 0.01% to 2% relative to AUG. Translation initiation from non-canonical start codons may contribute to the synthesis of peptides in both natural and synthetic biological systems

scholarly journals Naturally Occurring Adenines within mRNA Coding Sequences Affect Ribosome Binding and Expression in Escherichia coli

2006 ◽  
Vol 189 (2) ◽  
pp. 501-510 ◽  
Author(s):  
Jay E. Brock ◽  
Robert L. Paz ◽  
Patrick Cottle ◽  
Gary R. Janssen
Keyword(s):  
Escherichia Coli ◽  
Translation Initiation ◽  
Start Codon ◽  
Coding Sequences ◽  
E Coli ◽  
Ribosome Binding ◽  
Naturally Occurring ◽  
In Vivo Expression

ABSTRACT Translation initiation requires the precise positioning of a ribosome at the start codon. The major signals of bacterial mRNA that direct the ribosome to a translational start site are the Shine-Dalgarno (SD) sequence within the untranslated leader and the start codon. Evidence for the presence of many non-SD-led genes in prokaryotes provides a motive for studying additional interactions between ribosomes and mRNA that contribute to translation initiation. A high incidence of adenines has been reported downstream of the start codon for many Escherichia coli genes, and addition of downstream adenine-rich sequences increases expression from several genes in E. coli. Here we describe site-directed mutagenesis of the E. coli aroL, pncB, and cysJ coding sequences that was used to assess the contribution of naturally occurring adenines to in vivo expression and in vitro ribosome binding from mRNAs with different SD-containing untranslated leaders. Base substitutions that decreased the downstream adenines by one or two nucleotides decreased expression significantly from aroL-, pncB-, and cysJ-lacZ fusions; mutations that increased downstream adenines by one or two nucleotides increased expression significantly from aroL- and cysJ-lacZ fusions. Using primer extension inhibition (toeprint) and filter binding assays to measure ribosome binding, the changes in in vivo expression correlated closely with changes in in vitro ribosome binding strength. Our data are consistent with a model in which downstream adenines influence expression through their effects on the mRNA-ribosome association rate and the amount of ternary complex formed. This work provides evidence that adenine-rich sequence motifs might serve as a general enhancer of E. coli translation.

scholarly journals A Complementary Mechanism of Bacterial mRNA Translation Inhibition by Tetracyclines

2021 ◽  
Vol 12 ◽  
Author(s):  
Victor Barrenechea ◽  
Maryhory Vargas-Reyes ◽  
Miguel Quiliano ◽  
Pohl Milón
Keyword(s):  
Protein Synthesis ◽  
Translation Initiation ◽  
Ribosomal Subunit ◽  
Mrna Translation ◽  
Resistance Mechanisms ◽  
Dissociation Rate ◽  
Initiation Factor ◽  
Translation Elongation ◽  
Initiation Phase ◽  
Complementary Mechanism

Tetracycline has positively impacted human health as well as the farming and animal industries. Its extensive usage and versatility led to the spread of resistance mechanisms followed by the development of new variants of the antibiotic. Tetracyclines inhibit bacterial growth by impeding the binding of elongator tRNAs to the ribosome. However, a small number of reports indicated that Tetracyclines could also inhibit translation initiation, yet the molecular mechanism remained unknown. Here, we use biochemical and computational methods to study how Oxytetracycline (Otc), Demeclocycline (Dem), and Tigecycline (Tig) affect the translation initiation phase of protein synthesis. Our results show that all three Tetracyclines induce Initiation Factor IF3 to adopt a compact conformation on the 30S ribosomal subunit, similar to that induced by Initiation Factor IF1. This compaction was faster for Tig than Dem or Otc. Furthermore, all three tested tetracyclines affected IF1-bound 30S complexes. The dissociation rate constant of IF1 in early 30S complexes was 14-fold slower for Tig than Dem or Otc. Late 30S initiation complexes (30S pre-IC or IC) exhibited greater IF1 stabilization by Tig than for Dem and Otc. Tig and Otc delayed 50S joining to 30S initiation complexes (30S ICs). Remarkably, the presence of Tig considerably slowed the progression to translation elongation and retained IF1 in the resulting 70S initiation complex (70S IC). Molecular modeling of Tetracyclines bound to the 30S pre-IC and 30S IC indicated that the antibiotics binding site topography fluctuates along the initiation pathway. Mainly, 30S complexes show potential contacts between Dem or Tig with IF1, providing a structural rationale for the enhanced affinity of the antibiotics in the presence of the factor. Altogether, our data indicate that Tetracyclines inhibit translation initiation by allosterically perturbing the IF3 layout on the 30S, retaining IF1 during 70S IC formation, and slowing the transition toward translation elongation. Thus, this study describes a new complementary mechanism by which Tetracyclines may inhibit bacterial protein synthesis.

Effect of Tannins on the In Vitro Growth of Escherichia coli O157:H7 and In Vivo Growth of Generic Escherichia coli Excreted from Steers

2007 ◽  
Vol 70 (3) ◽  
pp. 543-550 ◽  
Author(s):  
BYENG R. MIN ◽  
WILLIAM E. PINCHAK ◽  
ROBIN C. ANDERSON ◽  
TODD R. CALLAWAY
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Bacterial Growth ◽  
Escherichia Coli O157 ◽  
Tannin Extract ◽  
Bacterial Growth Rate ◽  
Linear Effect ◽  
E Coli

The effect of commercially available chestnut and mimosa tannins in vitro (experiment 1) or in vivo (experiment 2) on the growth or recovery of Escherichia coli O157:H7 or generic fecal E. coli was evaluated. In experiment 1, the mean growth rate of E. coli O157:H7, determined via the measurement of optical density at 600 nm during anaerobic culture in tryptic soy broth at 37°C, was reduced (P < 0.05) with as little as 400 μg of either tannin extract per ml of culture fluid. The addition of 200, 400, 600, 800, and 1,200 μg of tannins per ml significantly (P < 0.01) reduced the specific bacterial growth rate when compared with the nontannin control. The specific growth rate decreased with increasing dose levels up to 800 μg of tannins per ml. Bacterial growth inhibition effects in chestnut tannins were less pronounced than in mimosa tannins. Chestnut tannin extract addition ranged from 0 to 1,200 μg/ml, and a linear effect (P < 0.05) was observed in cultures incubated for 6 h against the recovery of viable cells, determined via the plating of each strain onto MacConkey agar, of E. coli O157:H7 strains 933 and 86-24, but not against strain 6058. Similar tests with mimosa tannin extract showed a linear effect (P < 0.05) against the recovery of E. coli O157:H7 strain 933 only. The bactericidal effect observed in cultures incubated for 24 h with the tannin preparations was similar, although it was less than that observed from cultures incubated for 6 h. When chestnut tannins (15 g of tannins per day) were infused intraruminally to steers fed a Bermuda grass hay diet in experiment 2, fecal E. coli shedding was lower on days 3 (P < 0.03), 12 (P = 0.08), and 15 (P < 0.001) when compared with animals that were fed a similar diet without tannin supplementation. It was concluded that dietary levels and sources of tannins potentially reduce the shedding of E. coli from the gastrointestinal tract.

scholarly journals A Toxic Environment: a Growing Understanding of How Microbial Communities Affect Escherichia coli O157:H7 Shiga Toxin Expression

2020 ◽  
Vol 86 (24) ◽  
Author(s):  
Erin M. Nawrocki ◽  
Hillary M. Mosso ◽  
Edward G. Dudley
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Microbial Interactions ◽  
Severe Illness ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Lambdoid Prophage

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) strains, including E. coli O157:H7, cause severe illness in humans due to the production of Shiga toxin (Stx) and other virulence factors. Because Stx is coregulated with lambdoid prophage induction, its expression is especially susceptible to environmental cues. Infections with Stx-producing E. coli can be difficult to model due to the wide range of disease outcomes: some infections are relatively mild, while others have serious complications. Probiotic organisms, members of the gut microbiome, and organic acids can depress Stx production, in many cases by inhibiting the growth of EHEC strains. On the other hand, the factors currently known to amplify Stx act via their effect on the stx-converting phage. Here, we characterize two interactive mechanisms that increase Stx production by O157:H7 strains: first, direct interactions with phage-susceptible E. coli, and second, indirect amplification by secreted factors. Infection of susceptible strains by the stx-converting phage can expand the Stx-producing population in a human or animal host, and phage infection has been shown to modulate virulence in vitro and in vivo. Acellular factors, particularly colicins and microcins, can kill O157:H7 cells but may also trigger Stx expression in the process. Colicins, microcins, and other bacteriocins have diverse cellular targets, and many such molecules remain uncharacterized. The identification of additional Stx-amplifying microbial interactions will improve our understanding of E. coli O157:H7 infections and help elucidate the intricate regulation of pathogenicity in EHEC strains.

scholarly journals In Vivo Bioluminescent Monitoring of Therapeutic Efficacy and Pharmacodynamic Target Assessment of Antofloxacin against Escherichia coli in a Neutropenic Murine Thigh Infection Model

2017 ◽  
Vol 62 (1) ◽  
Author(s):  
Yu-Feng Zhou ◽  
Meng-Ting Tao ◽  
Yu-Zhang He ◽  
Jian Sun ◽  
Ya-Hong Liu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Subcutaneous Administration ◽  
Free Drug ◽  
Infection Model ◽  
Bioluminescent Imaging ◽  
Time Curve ◽  
Content Type ◽  
E Coli ◽  
Tract Infections

ABSTRACT Antimicrobial resistance among uropathogens has increased the rates of infection-related morbidity and mortality. Antofloxacin is a novel fluoroquinolone with broad-spectrum antibacterial activity against urinary Gram-negative bacilli, such as Escherichia coli. This study monitored the in vivo efficacy of antofloxacin using bioluminescent imaging and determined pharmacokinetic (PK)/pharmacodynamic (PD) targets against E. coli isolates in a neutropenic murine thigh infection model. The PK properties were determined after subcutaneous administration of antofloxacin at 2.5, 10, 40, and 160 mg/kg of body weight. Following thigh infection, the mice were treated with 2-fold-increasing doses of antofloxacin from 2.5 to 80 mg/kg administered every 12 h. Efficacy was assessed by quantitative determination of the bacterial burdens in thigh homogenates and was compared with the bioluminescent density. Antofloxacin demonstrated both static and killing endpoints in relation to the initial burden against all study strains. The PK/PD index area under the concentration-time curve (AUC)/MIC correlated well with efficacy (R 2 = 0.92), and the dose-response relationship was relatively steep, as observed with escalating doses of antofloxacin. The mean free drug AUC/MIC targets necessary to produce net bacterial stasis and 1-log10 and 2-log10 kill for each isolate were 38.7, 66.1, and 147.0 h, respectively. In vivo bioluminescent imaging showed a rapid decrease in the bioluminescent density at free drug AUC/MIC exposures that exceeded the stasis targets. The integration of these PD targets combined with the results of PK studies with humans will be useful in setting optimal dosing regimens for the treatment of urinary tract infections due to E. coli.

scholarly journals Coupled Release of Eukaryotic Translation Initiation Factors 5B and 1A from 80S Ribosomes following Subunit Joining

2007 ◽  
Vol 27 (6) ◽  
pp. 2384-2397 ◽  
Author(s):  
Jeanne M. Fringer ◽  
Michael G. Acker ◽  
Christie A. Fekete ◽  
Jon R. Lorsch ◽  
Thomas E. Dever
Keyword(s):  
Translation Initiation ◽  
Ribosomal Subunit ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation ◽  
Cell Extracts ◽  
Eukaryotic Translation Initiation ◽  
Subunit Joining

ABSTRACT The translation initiation GTPase eukaryotic translation initiation factor 5B (eIF5B) binds to the factor eIF1A and catalyzes ribosomal subunit joining in vitro. We show that rapid depletion of eIF5B in Saccharomyces cerevisiae results in the accumulation of eIF1A and mRNA on 40S subunits in vivo, consistent with a defect in subunit joining. Substituting Ala for the last five residues in eIF1A (eIF1A-5A) impairs eIF5B binding to eIF1A in cell extracts and to 40S complexes in vivo. Consistently, overexpression of eIF5B suppresses the growth and translation initiation defects in yeast expressing eIF1A-5A, indicating that eIF1A helps recruit eIF5B to the 40S subunit prior to subunit joining. The GTPase-deficient eIF5B-T439A mutant accumulated on 80S complexes in vivo and was retained along with eIF1A on 80S complexes formed in vitro. Likewise, eIF5B and eIF1A remained associated with 80S complexes formed in the presence of nonhydrolyzable GDPNP, whereas these factors were released from the 80S complexes in assays containing GTP. We propose that eIF1A facilitates the binding of eIF5B to the 40S subunit to promote subunit joining. Following 80S complex formation, GTP hydrolysis by eIF5B enables the release of both eIF5B and eIF1A, and the ribosome enters the elongation phase of protein synthesis.

scholarly journals In Vivo mRNA Profiling of Uropathogenic Escherichia coli from Diverse Phylogroups Reveals Common and Group-Specific Gene Expression Profiles

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Piotr Bielecki ◽  
Uthayakumar Muthukumarasamy ◽  
Denitsa Eckweiler ◽  
Agata Bielecka ◽  
Sarah Pohl ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Specific Gene ◽  
Content Type ◽  
E Coli ◽  
Human Host ◽  
Tract Infections

ABSTRACTmRNA profiling of pathogens during the course of human infections gives detailed information on the expression levels of relevant genes that drive pathogenicity and adaptation and at the same time allows for the delineation of phylogenetic relatedness of pathogens that cause specific diseases. In this study, we used mRNA sequencing to acquire information on the expression ofEscherichia colipathogenicity genes during urinary tract infections (UTI) in humans and to assign the UTI-associatedE. coliisolates to different phylogenetic groups. Whereas thein vivogene expression profiles of the majority of genes were conserved among 21E. colistrains in the urine of elderly patients suffering from an acute UTI, the specific gene expression profiles of the flexible genomes was diverse and reflected phylogenetic relationships. Furthermore, genes transcribedin vivorelative to laboratory media included well-described virulence factors, small regulatory RNAs, as well as genes not previously linked to bacterial virulence. Knowledge on relevant transcriptional responses that drive pathogenicity and adaptation of isolates to the human host might lead to the introduction of a virulence typing strategy into clinical microbiology, potentially facilitating management and prevention of the disease.IMPORTANCEUrinary tract infections (UTI) are very common; at least half of all women experience UTI, most of which are caused by pathogenicEscherichia colistrains. In this study, we applied massive parallel cDNA sequencing (RNA-seq) to provide unbiased, deep, and accurate insight into the nature and the dimension of the uropathogenicE. coligene expression profile during an acute UTI within the human host. This work was undertaken to identify key players in physiological adaptation processes and, hence, potential targets for new infection prevention and therapy interventions specifically aimed at sabotaging bacterial adaptation to the human host.

scholarly journals Role of Urinary Cathelicidin LL-37 and Human β-Defensin 1 in Uncomplicated Escherichia coli Urinary Tract Infections

2014 ◽  
Vol 82 (4) ◽  
pp. 1572-1578 ◽  
Author(s):  
Karen L. Nielsen ◽  
Pia Dynesen ◽  
Preben Larsen ◽  
Lotte Jakobsen ◽  
Paal S. Andersen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Urinary Tract Infections ◽  
Adult Women ◽  
Content Type ◽  
E Coli ◽  
Innate Defense ◽  
High Level ◽  
Tract Infections

ABSTRACTCathelicidin (LL-37) and human β-defensin 1 (hBD-1) are important components of the innate defense in the urinary tract. The aim of this study was to characterize whether these peptides are important for developing uncomplicatedEscherichia coliurinary tract infections (UTIs). This was investigated by comparing urinary peptide levels of UTI patients during and after infection to those of controls, as well as characterizing the fecal flora of participants with respect to susceptibility to LL-37 andin vivovirulence. Forty-seven UTI patients and 50 controls who had never had a UTI were included. Participants were otherwise healthy, premenopausal, adult women. LL-37 MIC levels were compared for fecalE. coliclones from patients and controls and were also compared based on phylotypes (A, B1, B2, and D).In vivovirulence was investigated in the murine UTI model by use of selected fecal isolates from patients and controls. On average, UTI patients had significantly more LL-37 in urine during infection than postinfection, and patient LL-37 levels postinfection were significantly lower than those of controls. hBD-1 showed similar urine levels for UTI patients and controls. FecalE. coliisolates from controls had higher LL-37 susceptibility than fecal and UTIE. coliisolates from UTI patients.In vivostudies showed a high level of virulence of fecalE. coliisolates from both patients and controls and showed no difference in virulence correlated with the LL-37 MIC level. The results indicate that the concentration of LL-37 in the urinary tract and low susceptibility to LL-37 may increase the likelihood of UTI in a complex interplay between host and pathogen attributes.

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