scholarly journals Translation of a leaderless reporter is robust during exponential growth and well sustained during stress conditions in Mycobacterium tuberculosis

2021 ◽  
Author(s):  
A. D. Grabowska ◽  
N. Andreu ◽  
T. Cortes
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Translational Regulation ◽  
Molecular Mechanisms ◽  
Growth Arrest ◽  
Optimal Growth ◽  
Growth Conditions ◽  
Stress Conditions ◽  
Shine Dalgarno Sequence ◽  
Overall Ratio

Abstract Mycobacterium tuberculosis expresses a large number of leaderless mRNA transcripts; these lack the 5’ leader region, which usually contains the Shine-Dalgarno sequence required for translation initiation in bacteria. In M. tuberculosis, transcripts encoding proteins with secondary adaptive functions are predominantly leaderless and the overall ratio of leaderless to Shine-Dalgarno transcripts significantly increases during growth arrest, suggesting that leaderless translation might be important during persistence in the host. However, whether these two types of transcripts are translated with differing efficiencies during stress conditions that induce growth arrest and during optimal growth conditions, is unclear. Here, using bioluminescent reporter strains, we detect robust leaderless translation during exponential in vitro growth and we show that leaderless translation is more stable than Shine-Dalgarno translation during adaptation to stress conditions. Upon entrance into nutrient starvation and after nitric oxide exposure, leaderless translation is significantly less affected by the stress than Shine-Dalgarno translation. Similarly, during the early stages of infection of macrophages, the levels of leaderless translation are more stable than those of Shine-Dalgarno translation. These results suggest that leaderless translation may offer an advantage in the physiology of M. tuberculosis. Identification of the molecular mechanisms underlying this translational regulation may provide insights into persistent infection.

scholarly journals Translation of a Leaderless Reporter Is Robust During Exponential Growth and Well Sustained During Stress Conditions in Mycobacterium tuberculosis

2021 ◽  
Vol 12 ◽  
Author(s):  
Anna D. Grabowska ◽  
Nuria Andreu ◽  
Teresa Cortes
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Translational Regulation ◽  
Molecular Mechanisms ◽  
Growth Arrest ◽  
Optimal Growth ◽  
Growth Conditions ◽  
Stress Conditions ◽  
Pcr Analysis ◽  
Shine Dalgarno Sequence

Mycobacterium tuberculosis expresses a large number of leaderless mRNA transcripts; these lack the 5′ leader region, which usually contains the Shine–Dalgarno sequence required for translation initiation in bacteria. In M. tuberculosis, transcripts encoding proteins like toxin–antitoxin systems are predominantly leaderless and the overall ratio of leaderless to Shine–Dalgarno transcripts significantly increases during growth arrest, suggesting that leaderless translation might be important during persistence in the host. However, whether these two types of transcripts are translated with differing efficiencies during optimal growth conditions and during stress conditions that induce growth arrest, is unclear. Here, we have used the desA1 (Rv0824c) and desA2 (Rv1094) gene pair as representative for Shine–Dalgarno and leaderless transcripts in M. tuberculosis respectively; and used them to construct bioluminescent reporter strains. We detect robust leaderless translation during exponential in vitro growth, and we show that leaderless translation is more stable than Shine–Dalgarno translation during adaptation to stress conditions. These changes are independent from transcription, as transcription levels did not significantly change following quantitative real-time PCR analysis. Upon entrance into nutrient starvation and after nitric oxide exposure, leaderless translation is significantly less affected by the stress than Shine–Dalgarno translation. Similarly, during the early stages of infection of macrophages, the levels of leaderless translation are transiently more stable than those of Shine–Dalgarno translation. These results suggest that leaderless translation may offer an advantage in the physiology of M. tuberculosis. Identification of the molecular mechanisms underlying this translational regulation may provide insights into persistent infection.

Water and Temperature Stress Impact Fitness of Acetohydroxyacid Synthase–Inhibiting Herbicide-Resistant Populations of Eastern Black Nightshade (Solanum ptychanthum)

Weed Science ◽  
2011 ◽  
Vol 59 (3) ◽  
pp. 341-348 ◽  
Author(s):  
Jamshid Ashigh ◽  
François J. Tardif
Keyword(s):  
Seed Production ◽  
Optimal Growth ◽  
Growth Conditions ◽  
Stress Conditions ◽  
Acetohydroxyacid Synthase ◽  
Eastern Black Nightshade ◽  
Black Nightshade ◽  
Reproductive Ability ◽  
Temperature Regimes ◽  
The Impact

Many substitutions in the herbicide target enzyme acetohydroxyacid synthase (AHAS) confer whole-plant resistance and may reduce plant fitness. This study was done to determine the impact of different watering and temperature regimes on the germination, growth, and seed production of eastern black nightshade populations resistant (R) to AHAS inhibitors as conferred by an Ala205Val substitution in their AHAS. Growth and reproductive ability of four R and four susceptible (S) populations were determined in growth-cabinet and greenhouse studies. The R populations had lower total berry and viable seed production per plant than S under optimal conditions because of slower berry maturation. Seed production of both S and R populations decreased under lower or higher than optimal watering regimes; however, this reduction was more pronounced for the S populations so that seed production was comparable across S and R. The R populations had significantly higher germination and vegetative growth under cooler alternating temperature regimes. Although there were no differences between R and S plants under stress conditions, under optimal growth conditions, the Ala205Val substitution comes at a significant cost in eastern black nightshade. Under optimal growth conditions and in the absence of herbicide selection, S populations should eventually dominate over R; however, the lack of fitness differences under stress conditions could enhance the persistence of the R individuals.

scholarly journals Ribonucleotide Reduction in Mycobacterium tuberculosis: Function and Expression of Genes Encoding Class Ib and Class II Ribonucleotide Reductases

2003 ◽  
Vol 71 (11) ◽  
pp. 6124-6131 ◽  
Author(s):  
Stephanie S. Dawes ◽  
Digby F. Warner ◽  
Liana Tsenova ◽  
Juliano Timm ◽  
John D. McKinney ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Class Ii ◽  
Growth Conditions ◽  
Small Subunit ◽  
Reverse Transcription Pcr ◽  
Hypoxic Conditions ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Class Ib

ABSTRACT Mycobacterium tuberculosis, the causative agent of tuberculosis, possesses a class Ib ribonucleotide reductase (RNR), encoded by the nrdE and nrdF2 genes, in addition to a putative class II RNR, encoded by nrdZ. In this study we probed the relative contributions of these RNRs to the growth and persistence of M. tuberculosis. We found that targeted knockout of the nrdF2 gene could be achieved only in the presence of a complementing allele, confirming that this gene is essential under normal, in vitro growth conditions. This observation also implied that the alternate class Ib small subunit encoded by the nrdF1 gene is unable to substitute for nrdF2 and that the class II RNR, NrdZ, cannot substitute for the class Ib enzyme, NrdEF2. Conversely, a ΔnrdZ null mutant of M. tuberculosis was readily obtained by allelic exchange mutagenesis. Quantification of levels of nrdE, nrdF2, nrdF1, and nrdZ gene expression by real-time, quantitative reverse transcription-PCR with molecular beacons by using mRNA from aerobic and O2-limited cultures showed that nrdZ was significantly induced under microaerophilic conditions, in contrast to the other genes, whose expression was reduced by O2 restriction. However, survival of the ΔnrdZ mutant strain was not impaired under hypoxic conditions in vitro. Moreover, the lungs of B6D2/F1 mice infected with the ΔnrdZ mutant had bacterial loads comparable to those of lungs infected with the parental wild-type strain, which argues against the hypothesis that nrdZ plays a significant role in the virulence of M. tuberculosis in this mouse model.

Role of GADD153 (growth arrest- and DNA damage-inducible gene 153) in vascular smooth muscle cell apoptosis

2001 ◽  
Vol 100 (3) ◽  
pp. 275-281 ◽  
Author(s):  
Michiya IGASE ◽  
Takafumi OKURA ◽  
Michitsugu NAKAMURA ◽  
Yasunori TAKATA ◽  
Yutaka KITAMI ◽  
...  
Keyword(s):  
Dna Damage ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Molecular Mechanisms ◽  
Growth Arrest ◽  
Cell Contact ◽  
Inducible Gene

GADD153 (growth arrest- and DNA damage-inducible gene 153) is expressed at very low levels in growing cells, but is markedly induced in response to a variety of cellular stresses, including glucose deprivation, exposure to genotoxic agents and other growth-arresting situations. Forced expression of GADD153 induces cell cycle arrest in many types of cells. It is also reported that GADD153 is directly associated with apoptosis. Recently we have reported that platelet-derived growth factor (PDGF)-BB induces apoptosis in cultured vascular smooth muscle cells (VSMC), but only when 100% confluency is reached. These results suggested that cell–cell contact inhibition (cell growth arrest) may be a critical factor for induction of VSMC apoptosis by PDGF-BB. In the present study, we explored the role of GADD153, one of a number of growth-arrest-related gene products, in the molecular mechanisms of VSMC apoptosis in vitro and in vivo. GADD153 was markedly induced at both the mRNA and protein levels, in parallel with the induction of VSMC apoptosis, after treatment with PDGF-BB. Moreover, overexpression of GADD153 in VSMC significantly reduced cell viability and induced apoptosis. In the carotid artery balloon injury model in rats, GADD153 protein was expressed in apoptotic VSMC which were positively stained by in situ DNA labelling. These results demonstrate an important role for GADD153 in the molecular mechanisms of VSMC apoptosis.

scholarly journals Introduction to in vitro culture of Ginkgo biloba (Linnaeus, 1771)

2020 ◽  
Vol 203 (12) ◽  
pp. 43-49
Author(s):  
Varvara Bessonova ◽  
Ol'ga Cherepanova
Keyword(s):  
Ginkgo Biloba ◽  
Optimal Growth ◽  
Growth Conditions ◽  
Biologically Active ◽  
Future Research ◽  
Planting Material ◽  
Murashige Skoog ◽  
Young Leaves ◽  
High Proliferative Activity

Abstract. The purpose of this research was to introduce Ginkgo biloba into culture, to study the composition and properties of its biologically active compounds. Methods. We researched the optimal growth conditions for obtaining a viable tissue culture, such as: concentration of phytohormones and other organic and nonorganic substances in Murashige – Skoog medium and light hours. The effectiveness of the standard method of sodium hypochloride sterilization of young leaves and vegetative buds also was verified. As a result, of conducting the experiment we were able to grow a living callus from leaves of G. biloba. Based on this result we can conclude that these conditions are acceptable for high proliferative activity of the plant. We were studied the effect of phytohormones NAA, at a concentration of 0.5 ml and 6-BAP, at a concentration of 2.5 ml. Also, was selected the ideal planting material for callus production – young leaves that were more sensitive to treatment with hypochloride. This research serves as the foundation for future research not only for our laboratory, but also for other research groups. The callus can be used to clone specimens of G. bilobain greenhouses. It will be use to extract and study unique chemical compounds, such as ginkgolides, bilobalides and various terpenes, contained in the extract of plants of this group.

scholarly journals In silico drug discovery strategies identified ADMET properties of decoquinate RMB041 and its potential drug targets against Mycobacterium Tuberculosis

2021 ◽  
Author(s):  
Kirsten Elke Knoll ◽  
Mietha M. van der Walt ◽  
Du toit Loots
Keyword(s):  
Mycobacterium Tuberculosis ◽  
In Silico ◽  
Drug Targets ◽  
Cellular Response ◽  
Molecular Mechanisms ◽  
Antimicrobial Agents ◽  
Screening Methods ◽  
Discovery Studio ◽  
Optimal Drug

The highly adaptive cellular response of Mycobacterium tuberculosis to various antibiotics and the high costs for clinical trials, hampers the development of novel antimicrobial agents with improved efficacy and safety. Subsequently, in silico drug screening methods are more commonly being used for the discovery and development of drugs, and have been proven useful for predicting the pharmacokinetics, toxicities, and targets, of prospective new antimicrobial agents. In this investigation, we used a reversed target fishing approach to determine potential hit targets and their possible interactions between M. tuberculosis and decoquinate RMB041, a propitious new antituberculosis compound. Two of the thirteen identified targets, Cyp130 and BlaI, were strongly proposed as optimal drug-targets for dormant M. tuberculosis, of which the first showed the highest comparative binding affinity to decoquinate RMB041. The metabolic pathways associated to the selected target proteins were compared to previously published molecular mechanisms of decoquinate RMB041 against M. tuberculosis, whereby we confirmed disrupted metabolism of proteins, cell wall components, and DNA. We also described the steps within these pathways that are inhibited and elaborated on decoquinate RMB041's activity against dormant M. tuberculosis. This compound has previously showed promising in vitro safety and good oral bioavailability, which were both supported by this in silico study. The pharmacokinetic properties and toxicity of this compound were predicted and investigated using the online tools pkCSM and SwissADME, and Discovery Studio software, which furthermore supports previous safety and bioavailability characteristics of decoquinate RMB041 for use as an antimycobacterial medication.

scholarly journals An Integrative Study Showing the Adaptation to Sub-Optimal Growth Conditions of Natural Populations of Arabidopsis thaliana: A Focus on Cell Wall Changes

Cells ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 2249
Author(s):  
Harold Duruflé ◽  
Philippe Ranocha ◽  
Thierry Balliau ◽  
Michel Zivy ◽  
Cécile Albenne ◽  
...  
Keyword(s):  
Cell Wall ◽  
Molecular Mechanisms ◽  
Environmental Changes ◽  
Natural Populations ◽  
Optimal Growth ◽  
Growth Conditions ◽  
Plant Adaptation ◽  
Population Responses ◽  
Integrative Study ◽  
Developmental Responses

In the global warming context, plant adaptation occurs, but the underlying molecular mechanisms are poorly described. Studying natural variation of the model plant Arabidopsisthaliana adapted to various environments along an altitudinal gradient should contribute to the identification of new traits related to adaptation to contrasted growth conditions. The study was focused on the cell wall (CW) which plays major roles in the response to environmental changes. Rosettes and floral stems of four newly-described populations collected at different altitudinal levels in the Pyrenees Mountains were studied in laboratory conditions at two growth temperatures (22 vs. 15 °C) and compared to the well-described Col ecotype. Multi-omic analyses combining phenomics, metabolomics, CW proteomics, and transcriptomics were carried out to perform an integrative study to understand the mechanisms of plant adaptation to contrasted growth temperature. Different developmental responses of rosettes and floral stems were observed, especially at the CW level. In addition, specific population responses are shown in relation with their environment and their genetics. Candidate genes or proteins playing roles in the CW dynamics were identified and will deserve functional validation. Using a powerful framework of data integration has led to conclusions that could not have been reached using standard statistical approaches.

scholarly journals Rpb4p, a Subunit of RNA Polymerase II, Mediates mRNA Export during Stress

2003 ◽  
Vol 14 (7) ◽  
pp. 2744-2755 ◽  
Author(s):  
Marganit Farago ◽  
Tal Nahari ◽  
Christopher Hammel ◽  
Charles N. Cole ◽  
Mordechai Choder
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mrna Export ◽  
Optimal Growth ◽  
Cellular Responses ◽  
Growth Conditions ◽  
Stress Conditions ◽  
Major Mechanism ◽  
A Subunit

Changes in gene expression represent a major mechanism by which cells respond to stress. We and other investigators have previously shown that the yeast RNA polymerase II subunit Rpb4p is required for transcription under various stress conditions, but not under optimal growth conditions. Here we show that, in addition to its role in transcription, Rpb4p is also required for mRNA export, but only when cells are exposed to stress conditions. The roles of Rpb4p in transcription and in mRNA export can be uncoupled genetically by specific mutations in Rpb4p. Both functions of Rpb4p are required to maintain cell viability during stress. We propose that Rpb4p participates in the cellular responses to stress at the interface of the transcription and the export machineries.

scholarly journals Three Mycobacterium tuberculosis Rel Toxin-Antitoxin Modules Inhibit Mycobacterial Growth and Are Expressed in Infected Human Macrophages

2008 ◽  
Vol 191 (5) ◽  
pp. 1618-1630 ◽  
Author(s):  
Shaleen B. Korch ◽  
Heidi Contreras ◽  
Josephine E. Clark-Curtiss
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Protein Pair ◽  
Growth Arrest ◽  
Broth Culture ◽  
Human Macrophages ◽  
In Vivo Analysis ◽  
Mycobacterial Growth

ABSTRACT Mycobacterium tuberculosis protein pairs Rv1246c-Rv1247c, Rv2865-Rv2866, and Rv3357-Rv3358, here named RelBE, RelFG, and RelJK, respectively, were identified based on homology to the Escherichia coli RelBE toxin:antitoxin (TA) module. In this study, we have characterized each Rel protein pair and have established that they are functional TA modules. Overexpression of individual M. tuberculosis rel toxin genes relE, relG, and relK induced growth arrest in Mycobacterium smegmatis; a phenotype that was completely reversible by expression of their cognate antitoxin genes, relB, relF, and relJ, respectively. We also provide evidence that RelB and RelE interact directly, both in vitro and in vivo. Analysis of the genetic organization and regulation established that relBE, relFG, and relJK form bicistronic operons that are cotranscribed and autoregulated, in a manner unlike typical TA modules. RelB and RelF act as transcriptional activators, inducing expression of their respective promoters. However, RelBE, RelFG, and RelJK (together) repress expression to basal levels of activity, while RelJ represses promoter activity altogether. Finally, we have determined that all six rel genes are expressed in broth-grown M. tuberculosis, whereas relE, relF, and relK are expressed during infection of human macrophages. This is the first demonstration of M. tuberculosis expressing TA modules in broth culture and during infection of human macrophages.

scholarly journals Role of the kdpDE Regulatory Operon of Mycobacterium tuberculosis in Modulating Bacterial Growth in vitro

2021 ◽  
Vol 12 ◽  
Author(s):  
Moloko C. Cholo ◽  
Maborwa T. Matjokotja ◽  
Ayman G. Osman ◽  
Ronald Anderson
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Bacterial Growth ◽  
Deletion Mutant ◽  
Regulatory System ◽  
Growth Conditions ◽  
Knockout Mutant ◽  
Double Knockout ◽  
Uptake Efficiency ◽  
Rates Of Growth

Bacteria use K+-uptake transporters differentially for adaptation in varying growth conditions. In Mycobacterium tuberculosis, two K+-uptake systems, the Trk comprising the CeoB and CeoC proteins and the Kdp consisting of the two-component system (TCS), KdpDE and KdpFABC, have been characterized, but their selective utilization during bacterial growth has not been completely explored. In the current study, the roles of the M. tuberculosis KdpDE regulatory system alone and in association with the Trk transporters in bacterial growth were investigated by evaluating the growth of M. tuberculosis KdpDE-deletion and KdpDE/Trk (KT)-double knockout mutant strains in planktonic culture under standard growth conditions. The KT-double knockout mutant strain was first constructed using homologous recombination procedures and was evaluated together with the KdpDE-deletion mutant and the wild-type (WT) strains with respect to their rates of growth, K+-uptake efficiencies, and K+-transporter gene expression during planktonic growth. During growth at optimal K+ concentrations and pH levels, selective deletion of the TCS KdpDE (KdpDE-deletion mutant) led to attenuation of bacterial growth and an increase in bacterial K+-uptake efficiency, as well as dysregulated expression of the kdpFABC and trk genes. Deletion of both the KdpDE and the Trk systems (KT-double knockout) also led to severely attenuated bacterial growth, as well as an increase in bacterial K+-uptake efficiency. These results demonstrate that the KdpDE regulatory system plays a key role during bacterial growth by regulating K+ uptake via modulation of the expression and activities of both the KdpFABC and Trk systems and is important for bacterial growth possibly by preventing cytoplasmic K+ overload.

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