scholarly journals Synthesis of the novel transporter YdhC, is regulated by the YdhB transcription factor controlling adenosine and adenine uptake

2020 ◽  
Author(s):  
Irina A. Rodionova ◽  
Ye Gao ◽  
Anand Sastry ◽  
Reo Yoo ◽  
Dmitry A. Rodionov ◽  
...  
Keyword(s):  
Nitrogen Sources ◽  
Gene Arrangement ◽  
The Novel ◽  
Rna Seq ◽  
Gene Encoding ◽  
E Coli ◽  
Binding Sequence ◽  
Predicted Binding Site ◽  
Lysr Family ◽  
Mg1655 Strain

AbstractThe YdhB transcriptional factor, re-named here AdnB, homologous to the allantoin regulator, AllS, was shown to regulate ydhC gene expression in Escherichia coli, which is divergently transcribed from adnB, and this gene arrangement is conserved in many Protreobacteria. The predicted consensus DNA binding sequence for YdhB is also conserved in Entrobacterial genomes. RNA-seq data confirmed the activation predicted due to the binding of AdnB as shown by Chip-Exo results. Fluorescent polarization experiments revealed binding of YdhB to the predicted binding site upstream of ydhC in the presence of 0.35 mM adenine, but not in its absence. The E. coli MG1655, strain lacking the ydhB gene, showed a lower level of ydhC mRNA in cells grown in M9-glucose supplemented with 2 mM adenosine. Adenosine and adenine are products of purine metabolism and provide sources of ammonium for many organisms. They are utilized under nitrogen starvation conditions as single nitrogen sources. Deletion of either the ydhC or the ydhB gene leads to a substantially decreased growth rate for E. coli in minimal M9 medium with glycerol as the carbon source and adenosine or adenine as the single nitrogen source. The ydhC mutant showed increased resistance to Paromomycine, Sulfathiazole and Sulfamethohazole using Biolog plates. We provide evidence that YdhB, (a novel LysR family regulator) activates expression of the ydhC gene, encoding a novel adenosine/adenine transporter in E. coli. The YdhB binding consensus for different groups of Enterobacteria was predicted.

scholarly journals A novel transcription factor PunR and Nac are involved in purine and purine nucleoside transporter punC regulation in E. coli

2021 ◽  
Author(s):  
Irina Rodionova ◽  
Ye Gao ◽  
Anand Sastry ◽  
Ying Hefner ◽  
Reo Yoo ◽  
...  
Keyword(s):  
Growth Rate ◽  
Minimal Medium ◽  
Binding Motif ◽  
Nucleoside Transporter ◽  
Transporter Gene ◽  
Rna Seq ◽  
Knockout Mutant ◽  
Bacterial Genomes ◽  
E Coli ◽  
Lysr Family

Abstract Many genes in bacterial genomes are of unknown function, often referred to as y-genes. Recently, novel analytic methods have divided bacterial transcriptomes into independently modulated sets of genes (iModulons). Functionally annotated iModulons that contain y-genes lead to testable hypotheses to elucidate y-gene function. Inversely correlated expression of a putative transporter gene, ydhC, relative to purine biosynthetic genes, has led to the hypothesis that it encodes a purine-related transporter and revealed a LysR-family regulator, YdhB, with a predicted 23-bp palindromic binding motif. RNA-Seq analysis of a ydhB knockout mutant confirmed the YdhB-dependent activation of ydhC in the presence of adenosine. The deletion of either the ydhC or the ydhB gene led to a substantially decreased growth rate for E. coli in minimal medium with adenosine as the nitrogen source, as well as with inosine or guanosine. Taken together, we provide clear evidence that YdhB activates the expression of the ydhC gene that encodes a novel purine transporter in E. coli. We propose that the genes ydhB and ydhC be re-named as punR and punC, respectively.

An iron-regulated gene required for utilization of aerobactin as an exogenous siderophore in Vibrio parahaemolyticus

Microbiology ◽  
2003 ◽  
Vol 149 (5) ◽  
pp. 1217-1225 ◽  
Author(s):  
Tatsuya Funahashi ◽  
Tomotaka Tanabe ◽  
Hiroaki Aso ◽  
Hiroshi Nakao ◽  
Yoshio Fujii ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Vibrio Parahaemolyticus ◽  
Outer Membrane Receptor ◽  
Gene Encoding ◽  
E Coli ◽  
Iron Deficient ◽  
Insertional Inactivation ◽  
Titration Assay ◽  
Binding Sequence

A previous investigation using the Fur titration assay system showed that Vibrio parahaemolyticus possesses a gene encoding a protein homologous to IutA, the outer-membrane receptor for ferric aerobactin in Escherichia coli. In this study, a 5·6 kb DNA region from the V. parahaemolyticus WP1 genome was cloned and two entire genes, iutA and alcD homologues, were identified which are absent from Vibrio cholerae genomic sequences. The V. parahaemolyticus IutA and AlcD proteins share 43 % identity with the Escherichia coli IutA protein and 24 % identity with the Bordetella bronchiseptica AlcD protein of unknown function, respectively. Primer extension analysis revealed that the iutA gene is transcribed in response to low-iron availability from a putative promoter overlapped with a sequence resembling a consensus E. coli Fur-binding sequence. In agreement with the above finding, V. parahaemolyticus effectively utilized exogenously supplied aerobactin for growth under iron-limiting conditions. Moreover, insertional inactivation of iutA impaired growth in the presence of aerobactin and incapacitated the outer-membrane fraction from iron-deficient cells for binding 55Fe-labelled aerobactin. These results indicate that the V. parahaemolyticus iutA homologue encodes an outer-membrane protein which functions as the receptor for ferric aerobactin. Southern blot analysis revealed that the iutA homologues are widely distributed in clinical and environmental isolates of V. parahaemolyticus. However, additional genes required for ferric aerobactin transport across the inner membrane remain to be clarified.

scholarly journals Pathoadaptive Mutations That Enhance Virulence: Genetic Organization of the cadA Regions ofShigella spp

2001 ◽  
Vol 69 (12) ◽  
pp. 7471-7480 ◽  
Author(s):  
William A. Day ◽  
Reinaldo E. Fernández ◽  
Anthony T. Maurelli
Keyword(s):  
The Novel ◽  
Gene Encoding ◽  
E Coli ◽  
Novel Gene ◽  
Genetic Linkages ◽  
Important Pathway ◽  
K 12 ◽  
Pathoadaptive Mutations ◽  
Host Tissues

ABSTRACT Pathoadaptive mutations improve the fitness of pathogenic species by modification of traits that interfere with factors (virulence and ancestral) required for survival in host tissues. A demonstrated pathoadaptive mutation is the loss of lysine decarboxylase (LDC) expression in Shigella species that have evolved from LDC-expressing Escherichia coli. Previous studies demonstrated that the product of LDC activity, cadaverine, blocks the action of Shigella enterotoxins and that the gene encoding LDC, cadA, was abolished by large chromosomal deletions in each Shigella species. To better understand the nature and evolution of these pathoadaptive mutations, remnants of thecad region were sequenced from the fourShigella species. These analyses reveal novel gene arrangements in this region of the pathogens' chromosomes. Insertion sequences, a phage genome, and/or loci from different positions on the ancestral E. coli chromosome displaced the cadAlocus to form distinct genetic linkages that are unique to eachShigella species. Hybridization studies, using an E. coli K-12 microarray, indicated that the genes displaced to form the novel linkages still remain in the Shigella genomes. None of these novel gene arrangements were observed in representatives of all E. coli phylogenies. Collectively, these observations indicate that inactivation of the cadAantivirulence gene occurred independently in each Shigellaspecies. The convergent evolution of these pathoadaptive mutations demonstrates that, following evolution from commensal E. coli, strong pressures in host tissues selectedShigella clones with increased fitness and virulence through the loss of an ancestral trait (LDC). These observations strongly support the role of pathoadaptive mutation as an important pathway in the evolution of pathogenic organisms.

Identification of a Transcriptional Activator (ChnR) and a 6-Oxohexanoate Dehydrogenase (ChnE) in the Cyclohexanol Catabolic Pathway in Acinetobacter sp. Strain NCIMB 9871 and Localization of the Genes That Encode Them

1999 ◽  
Vol 65 (11) ◽  
pp. 5158-5162 ◽  
Author(s):  
Hiroaki Iwaki ◽  
Yoshie Hasegawa ◽  
Masahiro Teraoka ◽  
Tai Tokuyama ◽  
Hélène Bergeron ◽  
...  
Keyword(s):  
Gene Sequence ◽  
Expression System ◽  
Transcriptional Activator ◽  
Fold Increase ◽  
Gene Arrangement ◽  
Catabolic Pathway ◽  
Gene Encoding ◽  
Cyclohexanone Monooxygenase ◽  
E Coli ◽  
Molecular Masses

ABSTRACT We identified chnR, a gene encoding an AraC-XylS type of transcriptional activator that regulates the expression ofchnB, the structural gene for cyclohexanone monooxygenase (CHMO) in Acinetobacter sp. strain NCIMB 9871. The gene sequence of chnE, which encodes an NADP+-linked 6-oxohexanoate dehydrogenase, the enzyme catalyzing the fifth step of cyclohexanol degradation, was also determined. The gene arrangement ischnB-chnE-chnR. The predicted molecular masses of the three polypeptides were verified by radiolabeling by using the T7 expression system. Inducible expression of cloned chnB inEscherichia coli depended upon the presence ofchnR. A transcriptionalchnB::lacZ fusion experiment revealed that cyclohexanone induces chnB expression in E. coli, in which a 22-fold increase in activity was observed.

scholarly journals Identification of a transcription factor, PunR, that regulates the purine and purine nucleoside transporter punC in E. coli

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Irina A. Rodionova ◽  
Ye Gao ◽  
Anand Sastry ◽  
Ying Hefner ◽  
Hyun Gyu Lim ◽  
...  
Keyword(s):  
Growth Rate ◽  
Minimal Medium ◽  
Binding Motif ◽  
Nucleoside Transporter ◽  
Transporter Gene ◽  
Rna Seq ◽  
Knockout Mutant ◽  
Bacterial Genomes ◽  
E Coli ◽  
Lysr Family

AbstractMany genes in bacterial genomes are of unknown function, often referred to as y-genes. Recently, the analytic methods have divided bacterial transcriptomes into independently modulated sets of genes (iModulons). Functionally annotated iModulons that contain y-genes lead to testable hypotheses to elucidate y-gene function. The inversely correlated expression of a putative transporter gene, ydhC, relative to purine biosynthetic genes, has led to the hypothesis that it encodes a purine-related transporter and revealed a LysR-family regulator, YdhB, with a predicted 23-bp palindromic binding motif. RNA-Seq analysis of a ydhB knockout mutant confirmed the YdhB-dependent activation of ydhC in the presence of adenosine. The deletion of either the ydhC or the ydhB gene led to a substantially decreased growth rate for E. coli in minimal medium with adenosine, inosine, or guanosine as the nitrogen source. Taken together, we provide clear evidence that YdhB activates the expression of the ydhC gene that encodes a purine transporter in E. coli. We propose that the genes ydhB and ydhC be re-named as punR and punC, respectively.

Development of 96 Multiple Injection-GC-MS Technique and Its Application in Protein Engineering of Natural and Non-Natural Enzymatic Reactions

2019 ◽  
Author(s):  
Anja Knorrscheidt ◽  
Pascal Püllmann ◽  
Eugen Schell ◽  
Dominik Homann ◽  
Erik Freier ◽  
...  
Keyword(s):  
Microtiter Plate ◽  
Axial Ligand ◽  
Cell System ◽  
Enzymatic Reactions ◽  
The Novel ◽  
Internal Standards ◽  
E Coli ◽  
Enzyme Screening ◽  
Plate Analysis ◽  
Microtiter Plate Analysis

Directed evolution requires the screening of enzyme libraries in biological matrices. Available assays are mostly substrate or enzyme specific. Chromatographic techniques like LC and GC overcome this limitation, but require long analysis times. The herein developed multiple injections in a single experimental run (MISER) using GC coupled to MS allows the injection of samples every 33 s resulting in 96-well microtiter plate analysis within 50 min. This technique is implementable in any GC-MS system with autosampling. Since the GC-MS is far less prone to ion suppression than LCMS, no chromatographic separation is required. This allows the utilisation of an internal standards and the detection of main and side-product. To prove the feasibility of the system in enzyme screening, two libraries were assessed: i) YfeX library in an E. coli whole cell system for the carbene-transfer reaction on indole revealing the novel axial ligand tryptophan, ii) a library of 616 chimeras of fungal unspecific peroxygenase (UPO) in S. cerevisiae supernatant for hydroxylation of tetralin resulting in novel constructs. The data quality and representation are automatically assessed by a new R-script.

scholarly journals AP2M1 Supports TGF-β Signals to Promote Collagen Expression by Inhibiting Caveolin Expression

2021 ◽  
Vol 22 (4) ◽  
pp. 1639
Author(s):  
Saerom Lee ◽  
Ga-Eun Lim ◽  
Yong-Nyun Kim ◽  
Hyeon-Sook Koo ◽  
Jaegal Shim
Keyword(s):  
Rna Seq ◽  
Gene Encoding ◽  
Caveolin 1 ◽  
Collagen Expression ◽  
Disease States ◽  
Tumor Growth Factor ◽  
Complex Process ◽  
Collagen Protein ◽  
Complex Regulation ◽  
Collagen Genes

The extracellular matrix (ECM) is important for normal development and disease states, including inflammation and fibrosis. To understand the complex regulation of ECM, we performed a suppressor screening using Caenorhabditis elegans expressing the mutant ROL-6 collagen protein. One cuticle mutant has a mutation in dpy-23 that encodes the μ2 adaptin (AP2M1) of clathrin-associated protein complex II (AP-2). The subsequent suppressor screening for dpy-23 revealed the lon-2 mutation. LON-2 functions to regulate body size through negative regulation of the tumor growth factor-beta (TGF-β) signaling pathway responsible for ECM production. RNA-seq analysis showed a dominant change in the expression of collagen genes and cuticle components. We noted an increase in the cav-1 gene encoding caveolin-1, which functions in clathrin-independent endocytosis. By knockdown of cav-1, the reduced TGF-β signal was significantly restored in the dpy-23 mutant. In conclusion, the dpy-23 mutation upregulated cav-1 expression in the hypodermis, and increased CAV-1 resulted in a decrease of TβRI. Finally, the reduction of collagen expression including rol-6 by the reduced TGF-β signal influenced the cuticle formation of the dpy-23 mutant. These findings could help us to understand the complex process of ECM regulation in organism development and disease conditions.

scholarly journals A transcription-based mechanism for oncogenic β-catenin-induced lethality in BRCA1/2-deficient cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Rebecca A. Dagg ◽  
Gijs Zonderland ◽  
Emilia Puig Lombardi ◽  
Giacomo G. Rossetti ◽  
Florian J. Groelly ◽  
...  
Keyword(s):  
Dna Damage ◽  
Replication Fork ◽  
High Throughput Sequencing ◽  
Germline Mutations ◽  
Cdk Inhibitor ◽  
Rna Seq ◽  
Gene Encoding ◽  
Origin Firing ◽  
Replication Fork Progression ◽  
Transcriptional Alterations

AbstractBRCA1 or BRCA2 germline mutations predispose to breast, ovarian and other cancers. High-throughput sequencing of tumour genomes revealed that oncogene amplification and BRCA1/2 mutations are mutually exclusive in cancer, however the molecular mechanism underlying this incompatibility remains unknown. Here, we report that activation of β-catenin, an oncogene of the WNT signalling pathway, inhibits proliferation of BRCA1/2-deficient cells. RNA-seq analyses revealed β-catenin-induced discrete transcriptome alterations in BRCA2-deficient cells, including suppression of CDKN1A gene encoding the CDK inhibitor p21. This accelerates G1/S transition, triggering illegitimate origin firing and DNA damage. In addition, β-catenin activation accelerates replication fork progression in BRCA2-deficient cells, which is critically dependent on p21 downregulation. Importantly, we find that upregulated p21 expression is essential for the survival of BRCA2-deficient cells and tumours. Thus, our work demonstrates that β-catenin toxicity in cancer cells with compromised BRCA1/2 function is driven by transcriptional alterations that cause aberrant replication and inflict DNA damage.

scholarly journals Complex transcriptional regulation of the Saccharomyces cerevisiae CYB2 gene encoding cytochrome b2: CYP1(HAP1) activator binds to the CYB2 upstream activation site UAS1-B2.

1991 ◽  
Vol 11 (7) ◽  
pp. 3762-3772 ◽  
Author(s):  
T Lodi ◽  
B Guiard
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Regulatory Region ◽  
Transcriptional Regulator ◽  
Direct Analysis ◽  
Transcriptional Level ◽  
Glucose Fermentation ◽  
Gene Encoding ◽  
Dnase I Footprinting ◽  
Mrna Transcripts ◽  
Binding Sequence

Expression of the Saccharomyces cerevisiae gene encoding cytochrome b2 (EC 1.2.2.3), CYB2, was investigated by direct analysis of mRNA transcripts and by measurement of the expression of lacZ fused to the CYB2 control regions. These studies indicated that regulation of the CYB2 gene is subject to several metabolic controls at the transcriptional level: inhibition due to glucose fermentation, induction by lactate, and inhibition in anaerobiosis or in absence of heme biosynthesis. Furthermore, we have shown that the CYB2 promoter contains one cis negative regulatory region and two heme-dependent positive regions, one of which is controlled by the transcriptional regulator CYP1 (HAP1) which is involved in the modulation of the expression of several oxygen-regulated genes. The CYP1 (HAP1)-binding sequence was located by gel retardation and DNase I footprinting experiments and compared with the binding sequences previously characterized in detail (UAS1CYC1, UAS'CYP3 (CYC7), and UASCTT1).

scholarly journals Molecular Characterization of the Gene Encoding a New AmpC β-Lactamase in a Clinical Strain of Acinetobacter Genomic Species 3

2004 ◽  
Vol 48 (4) ◽  
pp. 1374-1378 ◽  
Author(s):  
Alejandro Beceiro ◽  
Lourdes Dominguez ◽  
Anna Ribera ◽  
Jordi Vila ◽  
Francisca Molina ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Molecular Characterization ◽  
Biochemical Properties ◽  
Clinical Strain ◽  
The Novel ◽  
Gene Encoding ◽  
Genomic Species ◽  
First Time

ABSTRACT A presumptive chromosomal cephalosporinase (pI, 9.0) from a clinical strain of Acinetobacter genomic species 3 (AG3) is reported. The nucleotide sequence of this β-lactamase shows for the first time the gene encoding an AmpC enzyme in AG3. In addition, the biochemical properties of the novel AG3 AmpC β-lactamase are reported

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