scholarly journals Pseudouridine-FreeEscherichia coliRibosomes

2017 ◽  
Vol 200 (4) ◽  
Author(s):  
Michael O'Connor ◽  
Margus Leppik ◽  
Jaanus Remme
Keyword(s):  
Cell Growth ◽  
Functional Role ◽  
Ribosome Biogenesis ◽  
Content Type ◽  
E Coli ◽  
Pseudouridine Synthase ◽  
Domains Of Life ◽  
And Function ◽  
Critical Regions ◽  
Dependent Mechanism

ABSTRACTPseudouridine (Ψ) is present at conserved, functionally important regions in the ribosomal RNAs (rRNAs) from all three domains of life. Little, however, is known about the functions of Ψ modifications in bacterial ribosomes. AnEscherichia colistrain has been constructed in which all seven rRNA Ψ synthases have been inactivated and whose ribosomes are devoid of all Ψs. Surprisingly, this strain displays only minor defects in ribosome biogenesis and function, and cell growth is only modestly affected. This is in contrast to a strong requirement for Ψ in eukaryotic ribosomes and suggests divergent roles for rRNA Ψ modifications in these two domains.IMPORTANCEPseudouridine (Ψ) is the most abundant posttranscriptional modification in RNAs. In the ribosome, Ψ modifications are typically located at conserved, critical regions, suggesting they play an important functional role. In eukarya and archaea, rRNAs are modified by a single pseudouridine synthase (PUS) enzyme, targeted to rRNA via a snoRNA-dependent mechanism, while bacteria use multiple stand-alone PUS enzymes. Disruption of Ψ modification of rRNA in eukarya seriously impairs ribosome function and cell growth. We have constructed anE. colimultiple deletion strain lacking all Ψ modifications in rRNA. In contrast to the equivalent eukaryotic mutants, theE. colistrain is only modestly affected in growth, decoding, and ribosome biogenesis, indicating a differential requirement for Ψ modifications in these two domains.

scholarly journals Identification and Characterization of Genes Required for 5-Hydroxyuridine Synthesis in Bacillus subtilis and Escherichia coli tRNA

2019 ◽  
Vol 201 (20) ◽  
Author(s):  
Charles T. Lauhon
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Genomic Context ◽  
Similar Reduction ◽  
Content Type ◽  
E Coli ◽  
Wobble Position ◽  
Fertile Ground ◽  
And Function

ABSTRACT In bacteria, tRNAs that decode 4-fold degenerate family codons and have uridine at position 34 of the anticodon are typically modified with either 5-methoxyuridine (mo5U) or 5-methoxycarbonylmethoxyuridine (mcmo5U). These modifications are critical for extended recognition of some codons at the wobble position. Whereas the alkylation steps of these modifications have been described, genes required for the hydroxylation of U34 to give 5-hydroxyuridine (ho5U) remain unknown. Here, a number of genes in Escherichia coli and Bacillus subtilis are identified that are required for wild-type (wt) levels of ho5U. The yrrMNO operon is identified in B. subtilis as important for the biosynthesis of ho5U. Both yrrN and yrrO are homologs to peptidase U32 family genes, which includes the rlhA gene required for ho5C synthesis in E. coli. Deletion of either yrrN or yrrO, or both, gives a 50% reduction in mo5U tRNA levels. In E. coli, yegQ was found to be the only one of four peptidase U32 genes involved in ho5U synthesis. Interestingly, this mutant shows the same 50% reduction in (m)cmo5U as that observed for mo5U in the B. subtilis mutants. By analyzing the genomic context of yegQ homologs, the ferredoxin YfhL is shown to be required for ho5U synthesis in E. coli to the same extent as yegQ. Additional genes required for Fe-S biosynthesis and biosynthesis of prephenate give the same 50% reduction in modification. Together, these data suggest that ho5U biosynthesis in bacteria is similar to that of ho5C, but additional genes and substrates are required for complete modification. IMPORTANCE Modified nucleotides in tRNA serve to optimize both its structure and function for accurate translation of the genetic code. The biosynthesis of these modifications has been fertile ground for uncovering unique biochemistry and metabolism in cells. In this work, genes that are required for a novel anaerobic hydroxylation of uridine at the wobble position of some tRNAs are identified in both Bacillus subtilis and Escherichia coli. These genes code for Fe-S cluster proteins, and their deletion reduces the levels of the hydroxyuridine by 50% in both organisms. Additional genes required for Fe-S cluster and prephenate biosynthesis and a previously described ferredoxin gene all display a similar reduction in hydroxyuridine levels, suggesting that still other genes are required for the modification.

scholarly journals Regulation of Ribosomal Protein OperonsrplM-rpsI,rpmB-rpmG, andrplU-rpmAat the Transcriptional and Translational Levels

2016 ◽  
Vol 198 (18) ◽  
pp. 2494-2502 ◽  
Author(s):  
Leonid V. Aseev ◽  
Ludmila S. Koledinskaya ◽  
Irina V. Boni
Keyword(s):  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
General Rule ◽  
Single Copy ◽  
Translation Initiation Region ◽  
Content Type ◽  
Regulatory Pathways ◽  
E Coli

ABSTRACTIt is widely assumed that in the best-characterized model bacteriumEscherichia coli, transcription units encoding ribosomal proteins (r-proteins) and regulation of their expression have been already well defined. However, transcription start sites for severalE. colir-protein operons have been established only very recently, so that information concerning the regulation of these operons at the transcriptional or posttranscriptional level is still missing. This paper describes for the first time thein vivoregulation of three r-protein operons,rplM-rpsI,rpmB-rpmG, andrplU-rpmA. The results demonstrate that transcription of all three operons is subject to ppGpp/DksA-dependent negative stringent control under amino acid starvation, in parallel with the rRNA operons. By using single-copy translational fusions with the chromosomallacZgene, we show here that at the translation level only one of these operons,rplM-rpsI, is regulated by the mechanism of autogenous repression involving the 5′ untranslated region (UTR) of the operon mRNA, whilerpmB-rpmGandrplU-rpmAare not subject to this type of regulation. This may imply that translational feedback control is not a general rule for modulating the expression ofE. colir-protein operons. Finally, we report that L13, a primary protein in 50S ribosomal subunit assembly, serves as a repressor ofrplM-rpsIexpressionin vivo, acting at a target within therplMtranslation initiation region. Thus, L13 represents a novel example of regulatory r-proteins in bacteria.IMPORTANCEIt is important to obtain a deeper understanding of the regulatory mechanisms responsible for coordinated and balanced synthesis of ribosomal components. In this paper, we highlight the major role of a stringent response in regulating transcription of three previously unexplored r-protein operons, and we show that only one of them is subject to feedback regulation at the translational level. Improved knowledge of the regulatory pathways controlling ribosome biogenesis may promote the development of novel antibacterial agents.

scholarly journals A prion accelerates proliferation at the expense of lifespan

2020 ◽  
Author(s):  
David M. Garcia ◽  
Edgar A. Campbell ◽  
Christopher M. Jakobson ◽  
Mitsuhiro Tsuchiya ◽  
Acadia DiNardo ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Cell Growth ◽  
Cell Size ◽  
Functional Role ◽  
Growth And Survival ◽  
Pseudouridine Synthase ◽  
Epigenetic State ◽  
Altered Protein ◽  
Fluctuating Environments ◽  
The Cost

ABSTRACTOrganisms often commit to one of two strategies: living fast and dying young or living slow and dying old. In fluctuating environments, however, switching between these two strategies could be advantageous. Lifespan is often inversely correlated with cell size and proliferation, which are both limited by protein synthesis. Here we report that a highly conserved RNA-modifying enzyme, the pseudouridine synthase Pus4/TruB, can act as a prion, endowing yeast with greater proliferation rates at the cost of a shortened lifespan. Cells harboring the prion can grow larger and exhibit altered protein synthesis. This epigenetic state, [BIG+] (better in growth), allows cells to heritably yet reversibly alter their translational program, leading to the differential expression of hundreds of proteins, including many that regulate proliferation and aging. Our data reveal a functional role for aggregation of RNA-modifying enzymes in driving heritable epigenetic states that transform cell growth and survival.

scholarly journals N-Glycosylation of Haloferax volcanii Flagellins Requires Known Agl Proteins and Is Essential for Biosynthesis of Stable Flagella

2012 ◽  
Vol 194 (18) ◽  
pp. 4876-4887 ◽  
Author(s):  
Manuela Tripepi ◽  
Jason You ◽  
Sevcan Temel ◽  
Özlem Önder ◽  
Dustin Brisson ◽  
...  
Keyword(s):  
Gradient Centrifugation ◽  
Haloferax Volcanii ◽  
Content Type ◽  
Glycosylation Sites ◽  
Cscl Density Gradient ◽  
Domains Of Life ◽  
Covalently Linked ◽  
Glycosylation Pathway ◽  
A Minor ◽  
And Function

ABSTRACTN-glycosylation, a posttranslational modification required for the accurate folding and stability of many proteins, has been observed in organisms of all domains of life. Although the haloarchaeal S-layer glycoprotein was the first prokaryotic glycoprotein identified, little is known about the glycosylation of other haloarchaeal proteins. We demonstrate here that the glycosylation ofHaloferax volcaniiflagellins requires archaeal glycosylation (Agl) components involved in S-layer glycosylation and that the deletion of anyHfx. volcaniiaglgene impairs its swimming motility to various extents. A comparison of proteins in CsCl density gradient centrifugation fractions from supernatants of wild-typeHfx. volcaniiand deletion mutants lacking the oligosaccharyltransferase AglB suggests that when the Agl glycosylation pathway is disrupted, cells lack stable flagella, which purification studies indicate consist of a major flagellin, FlgA1, and a minor flagellin, FlgA2. Mass spectrometric analyses of FlgA1 confirm that its three predicted N-glycosylation sites are modified with covalently linked pentasaccharides having the same mass as that modifying its S-layer glycoprotein. Finally, the replacement of any of three predicted N-glycosylated asparagines of FlgA1 renders cells nonmotile, providing direct evidence for the first time that the N-glycosylation of archaeal flagellins is critical for motility. These results provide insight into the role that glycosylation plays in the assembly and function ofHfx. volcaniiflagella and demonstrate thatHfx. volcaniiflagellins are excellent reporter proteins for the study of haloarchaeal glycosylation processes.

scholarly journals RelA Functionally Suppresses the Growth Defect Caused by a Mutation in the G Domain of the Essential Der Protein

2008 ◽  
Vol 190 (9) ◽  
pp. 3236-3243 ◽  
Author(s):  
Jihwan Hwang ◽  
Masayori Inouye
Keyword(s):  
Cell Growth ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Synthetic Activity ◽  
Growth Defect ◽  
Multicopy Plasmid ◽  
Ribosomal Subunits ◽  
E Coli ◽  
Binding Domains ◽  
Gtp Binding

ABSTRACT A unique bacterial GTPase, Der, containing two tandem GTP-binding domains, is essential for cell growth and plays a crucial role in a large ribosomal subunit in Escherichia coli. The depletion of Der resulted in accumulation of both large and small ribosomal subunits and also affected the stability of large ribosomal subunits. However, its exact cellular function still remains elusive. Previously, we have shown that two G domain mutants, DerN118D and DerN321D, cannot support cell growth at low temperatures, suggesting that both GTP-binding domains are indispensable. In this study, we show that both Der variants are defective in ribosome biogenesis. Genetic screening of an E. coli genomic library was performed to identify the genes which, when expressed from a multicopy plasmid, can restore the growth defect of the DerN321D mutant at restrictive temperatures. Among seven suppressors isolated, four were located at 62.7 min on the E. coli genomic map, and the gene responsible for the suppression of DerN321D was identified as the relA gene which encodes a ribosome-associated (p)ppGpp synthetase. The synthetic activity of RelA was found to be essential for its DerN321D suppressor activity. Overexpression of RelA in a suppressor strain did not affect the expression of DerN321D but suppressed the polysome defects caused by the DerN321D mutant. This is the first demonstration of suppression of impaired function of Der by a functional enzyme. A possible mechanism of the suppression of DerN321D by RelA overproduction is discussed.

scholarly journals Abbreviated Pathway for Biosynthesis of 2-Thiouridine in Bacillus subtilis

2015 ◽  
Vol 197 (11) ◽  
pp. 1952-1962 ◽  
Author(s):  
Katherine A. Black ◽  
Patricia C. Dos Santos
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Cysteine Desulfurase ◽  
Content Type ◽  
E Coli ◽  
Wobble Position ◽  
Lysine Trna ◽  
Domains Of Life

ABSTRACTThe 2-thiouridine (s2U) modification of the wobble position in glutamate, glutamine, and lysine tRNA molecules serves to stabilize the anticodon structure, improving ribosomal binding and overall efficiency of the translational process. Biosynthesis of s2U inEscherichia colirequires a cysteine desulfurase (IscS), a thiouridylase (MnmA), and five intermediate sulfur-relay enzymes (TusABCDE). TheE. coliMnmA adenylates and subsequently thiolates tRNA to form the s2U modification.Bacillus subtilislacks IscS and the intermediate sulfur relay proteins, yet its genome contains a cysteine desulfurase gene,yrvO, directly adjacent tomnmA. The genomic synteny ofyrvOandmnmAcombined with the absence of the Tus proteins indicated a potential functionality of these proteins in s2U formation. Here, we provide evidence that theB. subtilisYrvO and MnmA are sufficient for s2U biosynthesis. A conditionalB. subtilisknockout strain showed that s2U abundance correlates with MnmA expression, andin vivocomplementation studies inE. coliIscS- or MnmA-deficient strains revealed the competency of these proteins in s2U biosynthesis.In vitroexperiments demonstrated s2U formation by YrvO and MnmA, and kinetic analysis established a partnership between theB. subtilisproteins that is contingent upon the presence of ATP. Furthermore, we observed that the slow-growth phenotype ofE. coliΔiscSand ΔmnmAstrains associated with s2U depletion is recovered byB. subtilis yrvOandmnmA. These results support the proposal that the involvement of a devoted cysteine desulfurase, YrvO, in s2U synthesis bypasses the need for a complex biosynthetic pathway by direct sulfur transfer to MnmA.IMPORTANCEThe 2-thiouridine (s2U) modification of the wobble position in glutamate, glutamine, and lysine tRNA is conserved in all three domains of life and stabilizes the anticodon structure, thus guaranteeing fidelity in translation. The biosynthesis of s2U inEscherichia colirequires seven proteins: the cysteine desulfurase IscS, the thiouridylase MnmA, and five intermediate sulfur-relay enzymes (TusABCDE).Bacillus subtilisand most Gram-positive bacteria lack a complete set of biosynthetic components. Interestingly, themnmAcoding sequence is located adjacent toyrvO, encoding a cysteine desulfurase. In this work, we provide evidence that theB. subtilisYrvO is able to transfer sulfur directly to MnmA. Both proteins are sufficient for s2U biosynthesis in a pathway independent of the one used inE. coli.

scholarly journals The Intimin-Like Protein FdeC Is Regulated by H-NS and Temperature in Enterohemorrhagic Escherichia coli

2014 ◽  
Vol 80 (23) ◽  
pp. 7337-7347 ◽  
Author(s):  
Donna M. Easton ◽  
Luke P. Allsopp ◽  
Minh-Duy Phan ◽  
Danilo Gomes Moriel ◽  
Guan Kai Goh ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Culture Conditions ◽  
Enterohemorrhagic Escherichia Coli ◽  
Infection Model ◽  
Content Type ◽  
E Coli ◽  
Uremic Syndrome ◽  
Animal Hosts ◽  
And Function

ABSTRACTEnterohemorrhagicEscherichia coli(EHEC) is a Shiga-toxigenic pathogen capable of inducing severe forms of enteritis (e.g., hemorrhagic colitis) and extraintestinal sequelae (e.g., hemolytic-uremic syndrome). The molecular basis of colonization of human and animal hosts by EHEC is not yet completely understood, and an improved understanding of EHEC mucosal adherence may lead to the development of interventions that could disrupt host colonization. FdeC, also referred to by its IHE3034 locus tag ECOK1_0290, is an intimin-like protein that was recently shown to contribute to kidney colonization in a mouse urinary tract infection model. The expression of FdeC is tightly regulatedin vitro, and FdeC shows promise as a vaccine candidate against extraintestinalE. colistrains. In this study, we characterized the prevalence, regulation, and function offdeCin EHEC. We showed that thefdeCgene is conserved in both O157 and non-O157 EHEC and encodes a protein that is expressed at the cell surface and promotes biofilm formation under continuous-flow conditions in a recombinantE. colistrain background. We also identified culture conditions under which FdeC is expressed and showed that minor alterations of these conditions, such as changes in temperature, can significantly alter the level of FdeC expression. Additionally, we demonstrated that the transcription of thefdeCgene is repressed by the global regulator H-NS. Taken together, our data suggest a role for FdeC in EHEC when it grows at temperatures above 37°C, a condition relevant to its specialized niche at the rectoanal junctions of cattle.

scholarly journals Genomic and Metabolic Profiling of Nonulosonic Acids in Vibrionaceae Reveal Biochemical Phenotypes of Allelic Divergence in Vibrio vulnificus

2011 ◽  
Vol 77 (16) ◽  
pp. 5782-5793 ◽  
Author(s):  
Amanda L. Lewis ◽  
Jean-Bernard Lubin ◽  
Shilpa Argade ◽  
Natasha Naidu ◽  
Biswa Choudhury ◽  
...  
Keyword(s):  
Vibrio Vulnificus ◽  
Hot Spot ◽  
Gene Clusters ◽  
Human Pathogens ◽  
Environmental Isolates ◽  
Content Type ◽  
The Family ◽  
Domains Of Life ◽  
Biochemical Analyses ◽  
And Function

ABSTRACTNonulosonic acids (NulOs) encompass a large group of structurally diverse nine-carbon backbone α-keto sugars widely distributed among the three domains of life. Mammals express a specialized version of NulOs called sialic acids, which are displayed in prominent terminal positions of cell surface and secreted glycoconjugates. Within bacteria, the ability to synthesize NulOs has been demonstrated in a number of human pathogens and is phylogenetically widespread. Here we examine the distribution, diversity, evolution, and function of NulO biosynthesis pathways in members of the familyVibrionaceae. Among 27 species ofVibrionaceaeexamined at the genomic level, 12 species containednabgene clusters. We document examples of duplication, divergence, horizontal transfer, and recombination ofnabgene clusters in differentVibrionaceaelineages. Biochemical analyses, including mass spectrometry, confirmed that many species do, in fact, produce di-N-acetylated NulOs. A library of clinical and environmental isolates ofVibrio vulnificusserved as a model for further investigation ofnaballele genotypes and levels of NulO expression. The data show that lineage I isolates produce about 20-fold higher levels of NulOs than lineage II isolates. Moreover,nabgene alleles found in a subset ofV. vulnificusclinical isolates express 40-fold higher levels of NulOs thannaballeles associated with environmental isolates. Taken together, the data implicate the familyVibrionaceaeas a “hot spot” of NulO evolution and suggest that these molecules may have diverse roles in environmental persistence and/or animal virulence.

scholarly journals Light Stimuli and Circadian Clock Affect Neural Development in Drosophila melanogaster

2021 ◽  
Vol 9 ◽  
Author(s):  
Eleni Dapergola ◽  
Pamela Menegazzi ◽  
Thomas Raabe ◽  
Anna Hovhanyan
Keyword(s):  
Cell Growth ◽  
Circadian Clock ◽  
Neural Development ◽  
Ribosome Biogenesis ◽  
Neural Circuit ◽  
Protein Biosynthesis ◽  
Tor Signaling ◽  
Light Stimuli ◽  
And Function ◽  
Cell Growth And Proliferation

Endogenous clocks enable organisms to adapt cellular processes, physiology, and behavior to daily variation in environmental conditions. Metabolic processes in cyanobacteria to humans are under the influence of the circadian clock, and dysregulation of the circadian clock causes metabolic disorders. In mouse and Drosophila, the circadian clock influences translation of factors involved in ribosome biogenesis and synchronizes protein synthesis. Notably, nutrition signals are mediated by the insulin receptor/target of rapamycin (InR/TOR) pathways to regulate cellular metabolism and growth. However, the role of the circadian clock in Drosophila brain development and the potential impact of clock impairment on neural circuit formation and function is less understood. Here we demonstrate that changes in light stimuli or disruption of the molecular circadian clock cause a defect in neural stem cell growth and proliferation. Moreover, we show that disturbed cell growth and proliferation are accompanied by reduced nucleolar size indicative of impaired ribosomal biogenesis. Further, we define that light and clock independently affect the InR/TOR growth regulatory pathway due to the effect on regulators of protein biosynthesis. Altogether, these data suggest that alterations in InR/TOR signaling induced by changes in light conditions or disruption of the molecular clock have an impact on growth and proliferation properties of neural stem cells in the developing Drosophila brain.

scholarly journals Mass and Density Measurements of Live and Dead Gram-Negative and Gram-Positive Bacterial Populations

2014 ◽  
Vol 80 (12) ◽  
pp. 3622-3631 ◽  
Author(s):  
Christina L. Lewis ◽  
Caelli C. Craig ◽  
Andre G. Senecal
Keyword(s):  
Cell Growth ◽  
Optical Density ◽  
Total Mass ◽  
Pathogenic Bacteria ◽  
Live Cells ◽  
Mass Measurements ◽  
Content Type ◽  
Density Measurements ◽  
E Coli ◽  
Physical Features

ABSTRACTMonitoring cell growth and measuring physical features of food-borne pathogenic bacteria are important for better understanding the conditions under which these organisms survive and proliferate. To address this challenge, buoyant masses of live and deadEscherichia coliO157:H7 andListeria innocuawere measured using Archimedes, a commercially available suspended microchannel resonator (SMR). Cell growth was monitored with Archimedes by observing increased cell concentration and buoyant mass values of live growing bacteria. These growth data were compared to optical density measurements obtained with a Bioscreen system. We observed buoyant mass measurements with Archimedes at cell concentrations between 105and 108cells/ml, while growth was not observed with optical density measurements until the concentration was 107cells/ml. Buoyant mass measurements of live and dead cells with and without exposure to hydrogen peroxide stress were also compared; live cells generally had a larger buoyant mass than dead cells. Additionally, buoyant mass measurements were used to determine cell density and total mass for both live and dead cells. DeadE. colicells were found to have a larger density and smaller total mass than liveE. colicells. In contrast, density was the same for both live and deadL. innocuacells, while the total mass was greater for live than for dead cells. These results contribute to the ongoing challenge to further develop existing technologies used to observe cell populations at low concentrations and to measure unique physical features of cells that may be useful for developing future diagnostics.

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