AbrB, A REGULATOR OF STATIONARY PHASE-INDUCED GENES IN BACILLUS, NEGATIVELY AFFECTS TRANSCRIPTION OF THE tyc-OPERON BY DIRECT INTERACTION WITH THE PROMOTER DNA

ABSTRACT Acinetobacter species encounter cycles of feast and famine in nature. We show that populations of A cinetobacter baylyi strain ADP1 remain dynamic for 6 weeks in batch culture. We created a library of lacZ reporters inserted into SalI sites in the genome and then isolated 30 genes with lacZ insertions whose expression was induced by starvation during long-term stationary phase compared with their expression during exponential growth. The genes encode metabolic, gene expression, DNA maintenance, envelope, and conserved hypothetical proteins.

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Hypoxic Preconditioning and Tolerance via Hypoxia Inducible Factor (HIF) 1α-linked Induction of P450 2C11 Epoxygenase in Astrocytes

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/sj.jcbfm.9600085 ◽

2005 ◽

Vol 25 (8) ◽

pp. 939-948 ◽

Cited By ~ 73

Author(s):

Mingyue Liu ◽

Nabil J Alkayed

Keyword(s):

Cytochrome P450 ◽

Hypoxia Inducible Factor ◽

Direct Interaction ◽

Glucose Deprivation ◽

Northern Blotting ◽

Hypoxic Preconditioning ◽

Mobility Shift ◽

Nuclear Extracts ◽

Promoter Dna ◽

Cytochrome P450 2C11

The brain's adaptive response to ischemic preconditioning (IPC) is mediated in part via hypoxia inducible factor (HIF)-responsive genes. We previously showed that IPC induces cytochrome P450 2C11 expression in the brain, associated with protection from stroke. Cytochrome P450 2C11 is an arachidonic acid (AA) epoxygenase expressed in astrocytes, which metabolizes AA to epoxyeicosatrienoic acids (EETs). We tested the hypotheses that hypoxic preconditioning (HPC) induces 2C11 expression in astrocytes via HIF-1α, and that the P450 epoxygenase pathway contributes to enhanced astrocyte tolerance to ischemia-like injury induced by oxygen-glucose deprivation (OGD). Primary cultured astrocytes were incubated under normoxic or hypoxic conditions for 1, 3, 6, 24, or 48 h, and protein levels of P450 2C11 and HIF-1α were measured by Western blotting. Additionally, 2C11 mRNA was measured by Northern blotting, and binding of HIF-1α to 2C11 promoter was evaluated using electrophoretic mobility shift assay (EMSA) with 2C11 promoter DNA containing putative HIF-binding sites. Levels of 2C11 mRNA and protein were significantly increased starting at 3 and 6 h of hypoxia, respectively. The increase in 2C11 expression was preceded by an increase in HIF-1α protein at 1 h of hypoxia, and EMSA showed a specific and direct interaction between 2C11 promoter DNA and HIF-1α in nuclear extracts from astrocytes. HPC and EETs reduced astrocyte cell death, and P450 epoxygenase inhibition prevented protection by HPC. We conclude that HPC induces tolerance in astrocytes, at least in part, via HIF-1α-linked upregulation of P450 2C11.

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Toxin–antitoxin operon kacAT of Klebsiella pneumoniae is regulated by conditional cooperativity via a W-shaped KacA–KacT complex

Nucleic Acids Research ◽

10.1093/nar/gkz563 ◽

2019 ◽

Vol 47 (14) ◽

pp. 7690-7702 ◽

Cited By ~ 7

Author(s):

Hongliang Qian ◽

Hao Yu ◽

Peifei Li ◽

E Zhu ◽

Qingqing Yao ◽

...

Keyword(s):

Crystal Structure ◽

Klebsiella Pneumoniae ◽

Previous Analysis ◽

Molecular Model ◽

Direct Interaction ◽

Bacterial Toxin ◽

Multidrug Tolerance ◽

Negative Autoregulation ◽

Promoter Dna

AbstractBacterial toxin–antitoxin pairs play important roles in bacterial multidrug tolerance. Gcn5-related N-acetyltransferase (GNAT) toxins inhibit translation by acetylation of aminoacyl-tRNAs and are counteracted by direct contacts with cognate ribbon–helix–helix (RHH) antitoxins. Our previous analysis showed that the GNAT toxin KacT and RHH antitoxin KacA of Klebsiella pneumoniae form a heterohexamer in solution and that the complex interacts with the cognate promoter DNA, resulting in negative autoregulation of kacAT transcription. Here, we present the crystal structure of DNA-bound KacAT complex at 2.2 Å resolution. The crystal structure revealed the formation of a unique heterohexamer, KacT–KacA2–KacA2–KacT. The direct interaction of KacA and KacT involves a unique W-shaped structure with the two KacT molecules at opposite ends. Inhibition of KacT is achieved by the binding of four KacA proteins that preclude the formation of an active KacT dimer. The kacAT operon is auto-regulated and we present an experimentally supported molecular model proposing that the KacT:KacA ratio controls kacAT transcription by conditional cooperativity. These results yield a profound understanding of how transcription GNAT–RHH pairs are regulated.

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Activation by NarL at the Escherichia coli ogt promoter

Biochemical Journal ◽

10.1042/bcj20200408 ◽

2020 ◽

Vol 477 (15) ◽

pp. 2807-2820

Author(s):

Patcharawarin Ruanto ◽

David L. Chismon ◽

Joanne Hothersall ◽

Rita E. Godfrey ◽

David J. Lee ◽

...

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Response Regulator ◽

Direct Interaction ◽

Α Subunit ◽

E Coli ◽

Terminal Domain ◽

Dna Alkyltransferase ◽

Promoter Dna ◽

Transcript Initiation

The Escherichia coli NarX/NarL two-component response-regulator system regulates gene expression in response to nitrate ions and the NarL protein is a global transcription factor, which activates transcript initiation at many target promoters. One such target, the E. coli ogt promoter, which controls the expression of an O6-alkylguanine-DNA-alkyltransferase, is dependent on NarL binding to two DNA targets centred at positions −44.5 and −77.5 upstream from the transcript start. Here, we describe ogt promoter derivatives that can be activated solely by NarL binding either at position −44.5 or position −77.5. We show that NarL can also activate the ogt promoter when located at position −67.5. We present data to argue that NarL-dependent activation of transcript initiation at the ogt promoter results from a direct interaction between NarL and a determinant in the C-terminal domain of the RNA polymerase α subunit. Footprinting experiments show that, at the −44.5 promoter, NarL and the C-terminal domain of the RNA polymerase α subunit bind to opposite faces of promoter DNA, suggesting an unusual mechanism of transcription activation. Our work suggests new organisations for activator-dependent transcription at promoters and future applications for biotechnology.

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A Derived Network-Based Interferon-Related Signature of Human Macrophages Responding toMycobacterium tuberculosis

BioMed Research International ◽

10.1155/2014/713071 ◽

2014 ◽

Vol 2014 ◽

pp. 1-16 ◽

Cited By ~ 2

Author(s):

Kang Wu ◽

Hai Fang ◽

Liang-Dong Lyu ◽

Douglas B. Lowrie ◽

Ka-Wing Wong ◽

...

Keyword(s):

Network Analysis ◽

Binding Sites ◽

Direct Interaction ◽

Host Responses ◽

Human Macrophage ◽

Human Macrophages ◽

Transcriptional Signature ◽

Highly Expressed Genes ◽

Induced Genes ◽

Human Infections

Network analysis of transcriptional signature typically relies on direct interaction between two highly expressed genes. However, this approach misses indirect and biological relevant interactions through a third factor (hub). Here we determine whether a hub-based network analysis can select an improved signature subset that correlates with a biological change in a stronger manner than the original signature. We have previously reported an interferon-related transcriptional signature (THP1r2Mtb-induced) fromMycobacterium tuberculosis(M. tb)-infected THP-1 human macrophage. We selected hub-connected THP1r2Mtb-induced genes into the refined network signature TMtb-iNet and grouped the excluded genes into the excluded signature TMtb-iEx. TMtb-iNet retained the enrichment of binding sites of interferon-related transcription factors and contained relatively more interferon-related interacting genes when compared to THP1r2Mtb-induced signature. TMtb-iNet correlated as strongly as THP1r2Mtb-induced signature on a public transcriptional dataset of patients with pulmonary tuberculosis (PTB). TMtb-iNet correlated more strongly in CD4+and CD8+T cells from PTB patients than THP1r2Mtb-induced signature and TMtb-iEx. When TMtb-iNet was applied to data during clinical therapy of tuberculosis, it resulted in the most pronounced response and the weakest correlation. Correlation on dataset from patients with AIDS or malaria was stronger for TMtb-iNet, indicating an involvement of TMtb-iNet in these chronic human infections. Collectively, the significance of this work is twofold: (1) we disseminate a hub-based approach in generating a biologically meaningful and clinically useful signature; (2) using this approach we introduce a new network-based signature and demonstrate its promising applications in understanding host responses to infections.

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Modulation of muscle creatine kinase promoter activity by the inducible orphan nuclear receptor TIS1

Biochemical Journal ◽

10.1042/bj3210281 ◽

1997 ◽

Vol 321 (2) ◽

pp. 281-287 ◽

Cited By ~ 2

Author(s):

Wan-Lin YANG ◽

Robert W. LIM

Keyword(s):

Creatine Kinase ◽

Nuclear Receptor ◽

Reporter Gene ◽

Direct Interaction ◽

C2c12 Cells ◽

Orphan Nuclear Receptor ◽

Muscle Creatine Kinase ◽

Muscle Creatine ◽

Promoter Dna

TIS1, an inducible orphan nuclear receptor, was originally isolated as a tumour-promoter-inducible gene in mouse 3T3 cells and later shown to be induced by growth factors and other extracellular stimuli. We show here that TIS1 mRNA was expressed in proliferating C2C12 mouse skeletal muscle cells but that the level of TIS1 expression increased during muscle differentiation. Overexpression of TIS1 transactivated muscle creatine kinase (MCK) reporter genes containing as little as 80 bp of the proximal 5ƀ flanking region. In contrast, a promoterless TIS1 construct and a frameshift mutant TIS1 construct were unable to transactivate the MCK reporter gene. Moreover, the effect exerted by TIS1 appeared to be selective for the MCK promoter. Treatment of C2C12 cells with forskolin, which is known to induce TIS1 expression, also stimulated MCK reporter gene activity. Interestingly, in vitro translated TIS1 protein failed to bind to the MCK promoter region, suggesting that the transactivation effect of TIS1 may be mediated without direct interaction of the protein with the MCK promoter DNA. Collectively, these results suggest that changing levels of TIS1 may help to modulate the expression of MCK, and perhaps other muscle-specific genes, in response to physiological changes.

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Morphological Characterization of Mycobacteriophage R1

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100051281 ◽

1974 ◽

Vol 32 ◽

pp. 254-255

Author(s):

B. L. Soloff ◽

T. A. Rado

Keyword(s):

Carbon Source ◽

Stationary Phase ◽

Growth Phase ◽

Minimal Medium ◽

Morphological Characterization ◽

Logarithmic Growth Phase ◽

Complete Lysis ◽

Lysogenic Culture ◽

Logarithmic Growth

Mycobacteriophage R1 was originally isolated from a lysogenic culture of M. butyricum. The virus was propagated on a leucine-requiring derivative of M. smegmatis, 607 leu−, isolated by nitrosoguanidine mutagenesis of typestrain ATCC 607. Growth was accomplished in a minimal medium containing glycerol and glucose as carbon source and enriched by the addition of 80 μg/ ml L-leucine. Bacteria in early logarithmic growth phase were infected with virus at a multiplicity of 5, and incubated with aeration for 8 hours. The partially lysed suspension was diluted 1:10 in growth medium and incubated for a further 8 hours. This permitted stationary phase cells to re-enter logarithmic growth and resulted in complete lysis of the culture.

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Nutrient-regulated gene expression in eukaryotes

Biochemical Society Symposium ◽

10.1042/bss0730085 ◽

2006 ◽

Vol 73 ◽

pp. 85-96 ◽

Cited By ~ 8

Author(s):

Richard J. Reece ◽

Laila Beynon ◽

Stacey Holden ◽

Amanda D. Hughes ◽

Karine Rébora ◽

...

Keyword(s):

Gene Expression ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Control ◽

Direct Interaction ◽

Signalling Pathways ◽

A Cell ◽

One Step ◽

Higher Eukaryotes ◽

Regulated Gene Expression

The recognition of changes in environmental conditions, and the ability to adapt to these changes, is essential for the viability of cells. There are numerous well characterized systems by which the presence or absence of an individual metabolite may be recognized by a cell. However, the recognition of a metabolite is just one step in a process that often results in changes in the expression of whole sets of genes required to respond to that metabolite. In higher eukaryotes, the signalling pathway between metabolite recognition and transcriptional control can be complex. Recent evidence from the relatively simple eukaryote yeast suggests that complex signalling pathways may be circumvented through the direct interaction between individual metabolites and regulators of RNA polymerase II-mediated transcription. Biochemical and structural analyses are beginning to unravel these elegant genetic control elements.

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