Regulation of the icl1 gene encoding the major isocitrate lyase in Mycobacterium smegmatis

2021 ◽  
Author(s):  
Eon-Min Ko ◽  
Ju-Yeon Kim ◽  
Sujin Lee ◽  
Suhkmann Kim ◽  
Jihwan Hwang ◽  
...  
Keyword(s):  
Binding Sites ◽  
Mycobacterium Smegmatis ◽  
Isocitrate Lyase ◽  
Regulatory Elements ◽  
Receptor Protein ◽  
Upstream Region ◽  
Regulation Mechanism ◽  
Glyoxylate Shunt ◽  
Gene Encoding ◽  
Key Enzyme

Mycobacterium smegmatis has two isocitrate lyase (ICL) isozymes (MSMEG_0911 and MSMEG_3706). We demonstrated that ICL1 (MSMEG_0911) is the predominantly expressed ICL in M. smegmatis and plays a major role in growth on acetate or fatty acid as the sole carbon and energy source. Expression of the icl1 gene in M. smegmatis was demonstrated to be strongly upregulated during growth on acetate relative to that in M. smegmatis grown on glucose. Expression of icl1 was shown to be positively regulated by the RamB activator, and three RamB-binding sites (RamBS1, RamBS2, and RamBS3) were identified in the upstream region of icl1 using DNase I footprinting analysis. Succinyl-CoA was shown to increase the binding affinity of RamB to its binding sites and enable RamB to bind to RamBS2 that is the most important site for RamB-mediated induction of icl1 expression. These results suggest that succinyl-CoA serves as a coinducer molecule for RamB. Our study also showed that cAMP receptor protein (Crp1: MSMEG_6189) represses icl1 expression in M. smegmatis grown in the presence of glucose. Therefore, the strong induction of icl1 expression during growth on acetate as the sole carbon source relative to the weak expression of icl1 during growth on glucose is likely to result from combined effects of RamB-mediated induction of icl1 in the presence of acetate and Crp-mediated repression of icl1 in the presence of glucose. IMPORTANCE Carbon flux through the glyoxylate shunt has been suggested to affect virulence, persistence, and antibiotic resistance of Mycobacterium tuberculosis . Therefore, it is important to understand the precise mechanism underlying the regulation of the icl gene encoding the key enzyme of the glyoxylate shunt. Using Mycobacterium smegmatis , this study revealed the regulation mechanism underlying induction of icl1 expression in M. smegmatis when the glyoxylate shunt is required. The conservation of the cis - and trans -acting regulatory elements related to icl1 regulation in both M. smegmatis and M. tuberculosis implies that the similar regulatory mechanism operates for the regulation of icl1 expression in M. tuberculosis .

scholarly journals The NtcA-Dependent P1 Promoter Is Utilized for glnA Expression in N2-Fixing Heterocysts of Anabaena sp. Strain PCC 7120

2004 ◽  
Vol 186 (21) ◽  
pp. 7337-7343 ◽  
Author(s):  
Ana Valladares ◽  
Alicia M. Muro-Pastor ◽  
Antonia Herrero ◽  
Enrique Flores
Keyword(s):  
Binding Site ◽  
In Vitro Transcription ◽  
Upstream Region ◽  
Gene Encoding ◽  
Differentiated Cells ◽  
Multiple Promoters ◽  
Key Enzyme ◽  
Anabaena Sp ◽  
Pcc 7120

ABSTRACT Expression of the glnA gene encoding glutamine synthetase, a key enzyme in nitrogen metabolism, is subject to a variety of regulatory mechanisms in different organisms. In the filamentous, N2-fixing cyanobacterium Anabaena sp. strain PCC 7120, glnA is expressed from multiple promoters that generate several transcripts whose abundance is influenced by NtcA, the transcription factor exerting global nitrogen control in cyanobacteria. Whereas RNAI originates from a canonical NtcA-dependent promoter (P1) and RNAII originates from a σ70-type promoter (P2), RNAIV is influenced by NtcA but the corresponding promoter (P3) does not have the structure of NtcA-activated promoters. Using RNA isolated from Anabaena filaments grown under different nitrogen regimens, we observed, in addition to these transcripts, RNAV, which has previously been detected only in in vitro transcription assays and should originate from P4. However, in heterocysts, which are differentiated cells specialized in N2 fixation, RNAI was the almost exclusive glnA transcript. Analysis of P glnA ::lacZ fusions containing different fragments of the glnA upstream region confirmed that fragments carrying P1, P2, or P3 and P4 have the ability to promote transcription. Mutation of the NtcA-binding site in P1 eliminated P1-directed transcription and allowed increased use of P2. The NtcA-binding site in the P1 promoter and binding of NtcA to this site appear to be key factors in determining glnA gene expression in vegetative cells and heterocysts.

scholarly journals PREDetector 2.0: Online and Enhanced Version of the Prokaryotic Regulatory Elements Detector Tool

2016 ◽  
Author(s):  
Pierre Tocquin ◽  
Aymeric Naômé ◽  
Samuel Jourdan ◽  
Sinaeda Anderssen ◽  
Samuel Hiard ◽  
...  
Keyword(s):  
Binding Sites ◽  
Regulatory Networks ◽  
Regulatory Elements ◽  
Upstream Region ◽  
Genomic Context ◽  
Bacterial Genomes ◽  
Microbial Genomes ◽  
Dna Motif ◽  
Putative Transcription Factor ◽  
Public Repositories

ABSTRACTIn the era that huge numbers of microbial genomes are being released in the databases, it becomes increasingly important to rapidly mine genes as well as predict the regulatory networks that control their expression. To this end, we have developed an improved and online version of the PREDetector software aimed at identifying putative transcription factor-binding sites (TFBS) in bacterial genomes. The original philosophy of PREDetector 1.0 is maintained, i.e. to allow users to freely fix the DNA-motif screening parameters, and to provide a statistical means to estimate the reliability of the prediction output. This new version offers an interactive table as well as graphics to dynamically alter the main screening parameters with automatic update of the list of identified putative TFBS. PREDetector 2.0 also has the following additional options: (i) access to genome sequences from different databases, (ii) access to weight matrices from public repositories, (iii) visualization of the predicted hits in their genomic context, (iv) grouping of hits identified in the same upstream region, (v) possibility to store the performed jobs, and (vi) automated export of the results in various formats. PREDetector 2.0 is available at http://predetector.fsc.ulg.ac.be/.

scholarly journals Positive and negative regulatory elements in the upstream region of the rat Cu/Zn-superoxide dismutase gene

1999 ◽  
Vol 339 (2) ◽  
pp. 335-341 ◽  
Author(s):  
Mun Seog CHANG ◽  
Hae Yong YOO ◽  
Hyune Mo RHO
Keyword(s):  
Superoxide Dismutase ◽  
Transcription Factors ◽  
Binding Sites ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Upstream Region ◽  
Sod1 Gene ◽  
Mobility Shift ◽  
Heterologous Promoter ◽  
Natural Context

Cu/Zn-superoxide dismutase (SOD1) catalyses the dismutation of superoxide radicals and neutralizes the oxidative effects of various chemicals. Deletion analysis of the upstream region of the rat SOD1 gene revealed that the promoter contains a positive regulatory element (PRE) and a negative regulatory element (NRE), which encompass the regions from -576 to -412 and from -412 to -305 respectively from the site of initiation of transcription. These DNA elements showed enhancer and silencer activities respectively in the natural context and in a heterologous promoter system. Using an electrophoretic-mobility-shift assay and a supershift assay with a specific antibody, the cis-elements of the PRE and NRE were identified as binding sites for transcription factors Elk1 and YY1 (Ying-Yang 1) respectively. Consistent with the presumed roles of the PRE and NRE, Elk1 increased SOD1 gene transcription about 4–5-fold, whereas YY1 exerted a negative effect of about 6-fold. Mutations of the Elk1- and YY1-binding sites led to diminution and elevation respectively of transcriptional activities, both in the natural context and in heterologous promoter systems. These results suggest that the transcription factors Elk1 and YY1, binding in the PRE and NRE respectively, co-ordinate the expression of the SOD1 gene.

Inactivation of mshB, a key gene in the mycothiol biosynthesis pathway in Mycobacterium smegmatis

Microbiology ◽  
2003 ◽  
Vol 149 (5) ◽  
pp. 1341-1349 ◽  
Author(s):  
Mamta Rawat ◽  
Svetozar Kovacevic ◽  
Helen Billman-Jacobe ◽  
Yossef Av-Gay
Keyword(s):  
Bacterial Growth ◽  
Mycobacterium Smegmatis ◽  
Parent Strain ◽  
Stationary Phases ◽  
Growth Cycle ◽  
Biosynthesis Pathway ◽  
Phenotypic Analysis ◽  
Gene Encoding ◽  
Key Enzyme ◽  
Amide Hydrolase

The mshB gene encoding N-acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside deacetylase (MshB) is a key enzyme in mycothiol biosynthesis. Disruption of mshB in Mycobacterium smegmatis resulted in decreased production of mycothiol (5–10 % of the parent strain mc2155) but did not abolish mycothiol synthesis completely. Complementation of the MshB− mutants with the mshB gene resulted in increased mycothiol production towards the exponential and stationary phases of the bacterial growth cycle. These results suggest that another enzyme is capable of mycothiol biosynthesis by providing N-acetylglucosaminylinositol deacetylation activity in the absence of MshB. One of the candidate enzymes capable of carrying out such reactions is the MshB orthologue mycothiol amide hydrolase, MCA. However, epichromosomal expression of mca in the MshB− mutants did not restore mycothiol levels to the level of the parent strain. Unlike other mutants, which have little or no detectable levels of mycothiol, the MshB− mutant did not exhibit increased resistance to isoniazid. However, the MshB− mutant was resistant to ethionamide. Phenotypic analysis of other mutants lacking mycothiol revealed that MshA− mutants also exhibit ethionamide resistance but that a MshC−mutant was sensitive to ethionamide, suggesting that mycothiol or its early intermediates influence ethionamide activation.

scholarly journals Regulation Mechanism of thealdGene Encoding Alanine Dehydrogenase in Mycobacterium smegmatis and Mycobacterium tuberculosis by the Lrp/AsnC Family Regulator AldR

2015 ◽  
Vol 197 (19) ◽  
pp. 3142-3153 ◽  
Author(s):  
Ji-A Jeong ◽  
Jaekyung Hyun ◽  
Jeong-Il Oh
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Binding Sites ◽  
Mycobacterium Smegmatis ◽  
Quaternary Structure ◽  
Consensus Sequence ◽  
Underlying Mechanism ◽  
Cooperative Binding ◽  
Regulation Mechanism ◽  
Content Type ◽  
Alanine Dehydrogenase

ABSTRACTIn the presence of alanine, AldR, which belongs to the Lrp/AsnC family of transcriptional regulators and regulatesaldencoding alanine dehydrogenase inMycobacterium smegmatis, changes its quaternary structure from a homodimer to an octamer with an open-ring conformation. Four AldR-binding sites (O2, O1, O4, and O3) with a consensus sequence of GA/T-N2-NWW/WWN-N2-A/TC were identified upstream of theM. smegmatisaldgene by means of DNase I footprinting analysis. O2, O1, and O4 are required for the induction ofaldexpression by alanine, while O3 is directly involved in the repression ofaldexpression. In addition to O3, both O1 and O4 are also necessary for full repression ofaldexpression in the absence of alanine, due to cooperative binding of AldR dimers to O1, O4, and O3. Binding of a molecule of the AldR octamer to thealdcontrol region was demonstrated to require two AldR-binding sites separated by three helical turns between their centers and one additional binding site that is in phase with the two AldR-binding sites. The cooperative binding of AldR dimers to DNA requires three AldR-binding sites that are aligned with a periodicity of three helical turns. ThealdRgene is negatively autoregulated independently of alanine. Comparative analysis ofaldexpression ofM. smegmatisandMycobacterium tuberculosisin conjunction with sequence analysis of bothaldcontrol regions led us to suggest that the expression of thealdgenes in both mycobacterial species is regulated by the same mechanism.IMPORTANCEIn mycobacteria, alanine dehydrogenase (Ald) is the enzyme required both to utilize alanine as a nitrogen source and to grow under hypoxic conditions by maintaining the redox state of the NADH/NAD+pool. Expression of thealdgene was reported to be regulated by the AldR regulator that belongs to the Lrp/AsnC (feast/famine) family, but the underlying mechanism was unknown. This study revealed the regulation mechanism ofaldinMycobacterium smegmatisandMycobacterium tuberculosis. Furthermore, a generalized arrangement pattern ofcis-acting regulatory sites for Lrp/AsnC (feast/famine) family regulators is suggested in this study.

scholarly journals Integration Host Factor and Cyclic AMP Receptor Protein Are Required for TyrR-Mediated Activation of tpl inCitrobacter freundii

1998 ◽  
Vol 180 (23) ◽  
pp. 6173-6186 ◽  
Author(s):  
Qing Bai ◽  
Ronald L. Somerville
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Molecular Mechanisms ◽  
Integration Host Factor ◽  
Host Factor ◽  
Receptor Protein ◽  
Upstream Region ◽  
Dnase I Footprinting ◽  
Promoter Dna ◽  
Camp Receptor

ABSTRACT The tpl gene of Citrobacter freundiiencodes an enzyme that catalyzes the conversion ofl-tyrosine to phenol, pyruvate, and ammonia. This gene is known to be positively regulated by TyrR. The amplitude of regulation attributable to this transcription factor is at least 20-fold. Three TyrR binding sites, designated boxes A, B, and C, centered at coordinates −272.5, −158.5, and −49.5, respectively, were identified in the upstream region of the tpl promoter. The results of mutational experiments suggest that TyrR binds in cooperative fashion to these sites. The nonavailability of any TyrR site impairs transcription. Full TyrR-mediated activation of tplrequired integration host factor (IHF) and the cAMP receptor protein (CRP). By DNase I footprinting, it was shown that the IHF binding site is centered at coordinate −85 and that there are CRP binding sites centered at coordinates −220 and −250. Mutational alteration of the IHF binding site reduced the efficiency of the tpl promoter by at least eightfold. The proposed roles of CRP and IHF are to introduce bends into tpl promoter DNA between boxes A and B or B and C. Multimeric TyrR dimers were demonstrated by a chemical cross-linking method. The formation of hexameric TyrR increased whentpl DNA was present. The participation of both IHF and CRP in the activation of the tpl promoter suggests that molecular mechanisms quite different from those that affect other TyrR-activated promoters apply to this system. A model wherein TyrR, IHF, and CRP collaborate to regulate the expression of thetpl promoter is presented.

Cloning of the inhibin/activin βB subunit gene from the Booroola Merino sheep

1991 ◽  
Vol 6 (1) ◽  
pp. 87-93 ◽  
Author(s):  
R. J. Rodgers
Keyword(s):  
Amino Acid ◽  
Cyclic Amp ◽  
Binding Sites ◽  
Human Gene ◽  
Upstream Region ◽  
Subunit Gene ◽  
Gene Encoding ◽  
Response Elements ◽  
Merino Sheep ◽  
Fertility Gene

ABSTRACT The gene encoding the inhibin/activin βB subunit was isolated from a Booroola Merino ewe which had two copies of the unidentified fertility gene. Both the DNA sequence (>87%) and amino acid sequence (>93%) were highly homologous to those of the human and rat genes. The upstream region of the gene was similar to that of the human and rat genes, having SP1 binding sites and no TATA or CAAT boxes and, like that of the rat, but unlike that of the human gene, no cyclic AMP response elements, suggesting that the βB gene of sheep and rats is regulated differently from that of man.

Effect of taste receptor protein T1R3 on the development of islet tissue of the murine pancreas

2019 ◽  
Vol 484 (1) ◽  
pp. 117-120
Author(s):  
V. O. Murovets ◽  
E. A. Sozontov ◽  
T. G. Zachepilo
Keyword(s):  
Parent Strain ◽  
Gene Knockout ◽  
Taste Receptor ◽  
Morphological Characteristics ◽  
Sweet Taste ◽  
Pancreatic Tissue ◽  
Receptor Protein ◽  
Gene Encoding ◽  
Islet Tissue ◽  
Knockout Animals

Protein T1R3, the main subunit of sweet, as well as amino acid, taste receptor, is expressed in the epithelium of the tongue and gastro intestinal tract, in β–cells of the pancreas, hypothalamus, and numerous other organs. Recently, convincing witnesses of T1R3 involvement in control of carbohydrate and lipid metabolism, and control of production of incretines and insulin, have been determined. In the study on Tas1r3-gene knockout mouse strain and parent strain C57Bl/6J as control, priority data concerning the effect of T1R3 on the morphological characteristics of Langerhans islets in the pancreas, are obtained. In Tas1r3 knockout animals, it is found that the size of the islets and their density in pancreatic tissue are reduced, as compared to the parent strain. Additionally, a decrease of expression of active caspase-3 in islets of gene-knockouts is demonstrated. The obtained data show that the lack of a functional, gene encoding sweet-taste receptor protein causes a dystrophy of the islet tissue and associates to the development of pathological changes in the pancreas specific to type-2 diabetes and obesity in humans.

scholarly journals Arabidopsis thaliana Myb59 Gene Is Involved in the Response to Heterodera schachtii Infestation, and Its Overexpression Disturbs Regular Development of Nematode-Induced Syncytia

2021 ◽  
Vol 22 (12) ◽  
pp. 6450
Author(s):  
Anita Wiśniewska ◽  
Kamila Wojszko ◽  
Elżbieta Różańska ◽  
Klaudia Lenarczyk ◽  
Karol Kuczerski ◽  
...  
Keyword(s):  
Cell Metabolism ◽  
Gene Promoter ◽  
Promoter Sequence ◽  
Regulatory Elements ◽  
Heterodera Schachtii ◽  
Myb Transcription Factor ◽  
Parasitic Nematodes ◽  
Regulatory Sequences ◽  
Gene Encoding ◽  
Constitutive Overexpression

Transcription factors are proteins that directly bind to regulatory sequences of genes to modulate and adjust plants’ responses to different stimuli including biotic and abiotic stresses. Sedentary plant parasitic nematodes, such as beet cyst nematode, Heterodera schachtii, have developed molecular tools to reprogram plant cell metabolism via the sophisticated manipulation of genes expression, to allow root invasion and the induction of a sequence of structural and physiological changes in plant tissues, leading to the formation of permanent feeding sites composed of modified plant cells (commonly called a syncytium). Here, we report on the AtMYB59 gene encoding putative MYB transcription factor that is downregulated in syncytia, as confirmed by RT-PCR and a promoter pMyb59::GUS activity assays. The constitutive overexpression of AtMYB59 led to the reduction in A. thaliana susceptibility, as indicated by decreased numbers of developed females, and to the disturbed development of nematode-induced syncytia. In contrast, mutant lines with a silenced expression of AtMYB59 were more susceptible to this parasite. The involvement of ABA in the modulation of AtMYB59 gene transcription appears feasible by several ABA-responsive cis regulatory elements, which were identified in silico in the gene promoter sequence, and experimental assays showed the induction of AtMYB59 transcription after ABA treatment. Based on these results, we suggest that AtMYB59 plays an important role in the successful parasitism of H. schachtii on A. thaliana roots.

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