Biochemical and biological properties of cortexillin III, a component of Dictyostelium DGAP1–cortexillin complexes

Cortexillins I–III are members of the α-actinin/spectrin subfamily of Dictyostelium calponin homology proteins. Unlike recombinant cortexillins I and II, which form homodimers as well as heterodimers in vitro, we find that recombinant cortexillin III is an unstable monomer but forms more stable heterodimers when coexpressed in Escherichia coli with cortexillin I or II. Expressed cortexillin III also forms heterodimers with both cortexillin I and II in vivo, and the heterodimers complex in vivo with DGAP1, a Dictyostelium GAP protein. Binding of cortexillin III to DGAP1 requires the presence of either cortexillin I or II; that is, cortexillin III binds to DGAP1 only as a heterodimer, and the heterodimers form in vivo in the absence of DGAP1. Expressed cortexillin III colocalizes with cortexillins I and II in the cortex of vegetative amoebae, the leading edge of motile cells, and the cleavage furrow of dividing cells. Colocalization of cortexillin III and F-actin may require the heterodimer/DGAP1 complex. Functionally, cortexillin III may be a negative regulator of cell growth, cytokinesis, pinocytosis, and phagocytosis, as all are enhanced in cortexillin III–null cells.

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In Vivo Modulation of a DnaJ Homolog, CbpA, by CbpM

Journal of Bacteriology ◽

10.1128/jb.01757-06 ◽

2007 ◽

Vol 189 (9) ◽

pp. 3635-3638 ◽

Cited By ~ 14

Author(s):

Matthew R. Chenoweth ◽

Nancy Trun ◽

Sue Wickner

Keyword(s):

Escherichia Coli ◽

Cell Growth ◽

Stationary Phase ◽

Specific Inhibitor ◽

Vitro Activity ◽

In Vitro Activity ◽

E Coli ◽

Hsp70 Chaperone

ABSTRACT CbpA, an Escherichia coli DnaJ homolog, can function as a cochaperone for the DnaK/Hsp70 chaperone system, and its in vitro activity can be modulated by CbpM. We discovered that CbpM specifically inhibits the in vivo activity of CbpA, preventing it from functioning in cell growth and division. Furthermore, we have shown that CbpM interacts with CbpA in vivo during stationary phase, suggesting that the inhibition of activity is a result of the interaction. These results reveal that the activity of the E. coli DnaK system can be regulated in vivo by a specific inhibitor.

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Decrease of miR-19b-3p in Brain Microvascular Endothelial Cells Attenuates Meningitic Escherichia coli-Induced Neuroinflammation via TNFAIP3-Mediated NF-κB Inhibition

Pathogens ◽

10.3390/pathogens8040268 ◽

2019 ◽

Vol 8 (4) ◽

pp. 268 ◽

Cited By ~ 2

Author(s):

Amjad ◽

Yang ◽

Li ◽

Fu ◽

Yang ◽

...

Keyword(s):

Escherichia Coli ◽

Endothelial Cells ◽

Negative Regulator ◽

Microvascular Endothelial Cells ◽

Brain Microvascular Endothelial Cells ◽

E Coli ◽

Inflammatory Damage ◽

Microvascular Endothelial

Meningitic Escherichia coli can traverse the host’s blood–brain barrier (BBB) and induce severe neuroinflammatory damage to the central nervous system (CNS). During this process, the host needs to reasonably balance the battle between bacteria and brain microvascular endothelial cells (BMECs) to minimize inflammatory damage, but this quenching of neuroinflammatory responses at the BBB is unclear. MicroRNAs (miRNAs) are widely recognized as key negative regulators in many pathophysiological processes, including inflammatory responses. Our previous transcriptome sequencing revealed numbers of differential miRNAs in BMECs upon meningitic E. coli infection; we next sought to explore whether and how these miRNAs worked to modulate neuroinflammatory responses at meningitic E. coli entry of the BBB. Here, we demonstrated in vivo and in vitro that meningitic E. coli infection of BMECs significantly downregulated miR-19b-3p, which led to attenuated production of proinflammatory cytokines and chemokines via increasing the expression of TNFAIP3, a negative regulator of NF-κB signaling. Moreover, in vivo injection of miR-19b-3p mimics during meningitic E. coli challenge further aggravated the inflammatory damage to mice brains. These in vivo and in vitro findings indicate a novel quenching mechanism of the host by attenuating miR-19b-3p/TNFAIP3/NF-κB signaling in BMECs in response to meningitic E. coli, thus preventing CNS from further neuroinflammatory damage.

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Stimulation of the λ p R promoter by Escherichia coli SeqA protein requires downstream GATC sequences and involves late stages of transcription initiation

Microbiology ◽

10.1099/mic.0.29110-0 ◽

2006 ◽

Vol 152 (10) ◽

pp. 2985-2992 ◽

Cited By ~ 7

Author(s):

Robert Łyżeń ◽

Grzegorz Wȩgrzyn ◽

Alicja Wȩgrzyn ◽

Agnieszka Szalewska-Pałasz

Keyword(s):

Escherichia Coli ◽

Transcription Initiation ◽

Negative Regulator ◽

Regulation Of Transcription ◽

Chromosomal Dna ◽

Transcription Activator ◽

Transcription Analysis ◽

Stimulation Of

Escherichia coli SeqA protein is a major negative regulator of chromosomal DNA replication acting by sequestration, and thus inactivation, of newly formed oriC regions. However, other activities of this protein have been discovered recently, one of which is regulation of transcription. SeqA has been demonstrated to be a specific transcription factor acting at bacteriophage λ promoters p I, p aQ and p R. While SeqA-mediated stimulation of p I and p aQ occurs by facilitating functions of another transcription activator protein, cII, a mechanism for stimulation of p R remains largely unknown. Here, it has been demonstrated that two GATC sequences, located 82 and 105 bp downstream of the p R transcription start site, are necessary for this stimulation both in vivo and in vitro. SeqA-mediated activation of p R was as effective on a linear DNA template as on a supercoiled one, indicating that alterations in DNA topology are not likely to facilitate the SeqA effect. In vitro transcription analysis demonstrated that the most important regulatory effect of SeqA in p R transcription occurs after open complex formation, namely during promoter clearance. SeqA did not influence the appearance and level of abortive transcripts or the pausing during transcription elongation. Interestingly, SeqA is one of few known prokaryotic transcription factors which bind downstream of the regulated promoter and still act as transcription activators.

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Differential Mechanisms of Binding of Anti-Sigma Factors Escherichia coli Rsd and Bacteriophage T4 AsiA to E. coli RNA Polymerase Lead to Diverse Physiological Consequences

Journal of Bacteriology ◽

10.1128/jb.01792-07 ◽

2008 ◽

Vol 190 (10) ◽

pp. 3434-3443 ◽

Cited By ~ 24

Author(s):

Umender K. Sharma ◽

Dipankar Chatterji

Keyword(s):

Escherichia Coli ◽

Cell Growth ◽

Rna Polymerase ◽

Bacteriophage T4 ◽

Sigma Factors ◽

E Coli ◽

Yeast Two Hybrid System ◽

Vitro Binding

ABSTRACT Anti-sigma factors Escherichia coli Rsd and bacteriophage T4 AsiA bind to the essential housekeeping sigma factor, σ70, of E. coli. Though both factors are known to interact with the C-terminal region of σ70, the physiological consequences of these interactions are very different. This study was undertaken for the purpose of deciphering the mechanisms by which E. coli Rsd and bacteriophage T4 AsiA inhibit or modulate the activity of E. coli RNA polymerase, which leads to the inhibition of E. coli cell growth to different amounts. It was found that AsiA is the more potent inhibitor of in vivo transcription and thus causes higher inhibition of E. coli cell growth. Measurements of affinity constants by surface plasmon resonance experiments showed that Rsd and AsiA bind to σ70 with similar affinity. Data obtained from in vivo and in vitro binding experiments clearly demonstrated that the major difference between AsiA and Rsd is the ability of AsiA to form a stable ternary complex with RNA polymerase. The binding patterns of AsiA and Rsd with σ70 studied by using the yeast two-hybrid system revealed that region 4 of σ70 is involved in binding to both of these anti-sigma factors; however, Rsd interacts with other regions of σ70 as well. Taken together, these results suggest that the higher inhibition of E. coli growth by AsiA expression is probably due to the ability of the AsiA protein to trap the holoenzyme RNA polymerase rather than its higher binding affinity to σ70.

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Dual Function of Aar, a Member of the New AraC Negative Regulator Family, in Escherichia coli Gene Expression

Infection and Immunity ◽

10.1128/iai.00100-20 ◽

2020 ◽

Vol 88 (6) ◽

Cited By ~ 2

Author(s):

Abigail S. Mickey ◽

James P. Nataro

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Negative Regulator ◽

Dual Function ◽

Content Type ◽

Virulence Gene Expression ◽

E Coli ◽

K 12

ABSTRACT Enteroaggregative Escherichia coli (EAEC) is an E. coli pathotype associated with diarrhea and growth faltering. EAEC virulence gene expression is controlled by the autoactivated AraC family transcriptional regulator, AggR. AggR activates transcription of a large number of virulence genes, including Aar, which in turn acts as a negative regulator of AggR itself. Aar has also been shown to affect expression of E. coli housekeeping genes, including H-NS, a global regulator that acts at multiple promoters and silences AT-rich genes (such as those in the AggR regulon). Although Aar has been shown to bind both AggR and H-NS in vitro, functional significance of these interactions has not been shown in vivo. In order to dissect this regulatory network, we removed the complex interdependence of aggR and aar by placing the genes under the control of titratable promoters. We measured phenotypic and genotypic changes on downstream genes in EAEC strain 042 and E. coli K-12 strain DH5α, which lacks the AggR regulon. In EAEC, we found that low expression of aar increases aafA fimbrial gene expression via H-NS; however, when aar is more highly expressed, it acts as a negative regulator via AggR. In DH5α, aar affected expression of E. coli genes in some cases via H-NS and in some cases independent of H-NS. Our data support the model that Aar interacts in concert with AggR, H-NS, and possibly other regulators and that these interactions are likely to be functionally significant in vivo.

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Covariance of Complementary rRNA Loop Nucleotides Does Not Necessarily Represent Functional Pseudoknot Formation In Vivo

Journal of Bacteriology ◽

10.1128/jb.182.20.5671-5675.2000 ◽

2000 ◽

Vol 182 (20) ◽

pp. 5671-5675 ◽

Cited By ~ 2

Author(s):

Natalya S. Chernyaeva ◽

Emanuel J. Murgola

Keyword(s):

Escherichia Coli ◽

Protein Synthesis ◽

Cell Growth ◽

Site Directed Mutagenesis ◽

23S Rrna ◽

Directed Mutagenesis ◽

Bacterial Cells ◽

Domain I

ABSTRACT We examined mutationally a two-hairpin structure (nucleotides 57 to 70 and 76 to 110) in a region of domain I ofEscherichia coli 23S rRNA that has been implicated in specific functions in protein synthesis by other studies. On the basis of the observed covariance of several nucleotides in each loop inBacteria, Archaea, and chloroplasts, the two hairpins have been proposed to form a pseudoknot. Here, appropriate loop changes were introduced in vitro by site-directed mutagenesis to eliminate any possibility of base pairing between the loops. The bacterial cells containing each cloned mutant rRNA operon were then examined for cell growth, termination codon readthrough, and assembly of the mutant rRNAs into functional ribosomes. The results show that, under the conditions examined, the two hairpins do not form a pseudoknot structure that is required for the functioning of the ribosome in vivo and therefore that sequence covariance does not necessarily indicate the formation of a functional pseudoknot.

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The PspA Protein of Escherichia coli Is a Negative Regulator of ς54-Dependent Transcription

Journal of Bacteriology ◽

10.1128/jb.182.2.311-319.2000 ◽

2000 ◽

Vol 182 (2) ◽

pp. 311-319 ◽

Cited By ~ 71

Author(s):

Jonathan Dworkin ◽

Goran Jovanovic ◽

Peter Model

Keyword(s):

Escherichia Coli ◽

Sigma Factor ◽

Negative Regulator ◽

Regulatory Domain ◽

Expression Of Genes ◽

The Family ◽

Initiation Of Transcription ◽

Rna Polymerase Holoenzyme

ABSTRACT In Eubacteria, expression of genes transcribed by an RNA polymerase holoenzyme containing the alternate sigma factor ς54 is positively regulated by proteins belonging to the family of enhancer-binding proteins (EBPs). These proteins bind to upstream activation sequences and are required for the initiation of transcription at the ς54-dependent promoters. They are typically inactive until modified in their N-terminal regulatory domain either by specific phosphorylation or by the binding of a small effector molecule. EBPs lacking this domain, such as the PspF activator of the ς54-dependent pspA promoter, are constitutively active. We describe here the in vivo and in vitro properties of the PspA protein of Escherichia coli, which negatively regulates expression of the pspA promoter without binding DNA directly.

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Discovery and Characterization of Three New Escherichia coli Septal Ring Proteins That Contain a SPOR Domain: DamX, DedD, and RlpA

Journal of Bacteriology ◽

10.1128/jb.01244-09 ◽

2009 ◽

Vol 192 (1) ◽

pp. 242-255 ◽

Cited By ~ 57

Author(s):

S. J. Ryan Arends ◽

Kyle Williams ◽

Renada J. Scott ◽

Silvana Rolong ◽

David L. Popham ◽

...

Keyword(s):

Escherichia Coli ◽

Caulobacter Crescentus ◽

Negative Regulator ◽

Aquifex Aeolicus ◽

E Coli ◽

Division Site ◽

Genes Encoding

ABSTRACT SPOR domains are ∼70 amino acids long and occur in >1,500 proteins identified by sequencing of bacterial genomes. The SPOR domains in the FtsN cell division proteins from Escherichia coli and Caulobacter crescentus have been shown to bind peptidoglycan. Besides FtsN, E. coli has three additional SPOR domain proteins—DamX, DedD, and RlpA. We show here that all three of these proteins localize to the septal ring in E. coli. The loss of DamX or DedD either alone or in combination with mutations in genes encoding other division proteins resulted in a variety of division phenotypes, demonstrating that DamX and DedD participate in cytokinesis. In contrast, RlpA mutants divided normally. Follow-up studies revealed that the SPOR domains themselves localize to the septal ring in vivo and bind peptidoglycan in vitro. Even SPOR domains from heterologous organisms, including Aquifex aeolicus, localized to septal rings when produced in E. coli and bound to purified E. coli peptidoglycan sacculi. We speculate that SPOR domains localize to the division site by binding preferentially to septal peptidoglycan. We further suggest that SPOR domain proteins are a common feature of the division apparatus in bacteria. DamX was characterized further and found to interact with multiple division proteins in a bacterial two-hybrid assay. One interaction partner is FtsQ, and several synthetic phenotypes suggest that DamX is a negative regulator of FtsQ function.

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MDM1 is a microtubule-binding protein that negatively regulates centriole duplication

Molecular Biology of the Cell ◽

10.1091/mbc.e15-04-0235 ◽

2015 ◽

Vol 26 (21) ◽

pp. 3788-3802 ◽

Cited By ~ 10

Author(s):

Daniel Van de Mark ◽

Dong Kong ◽

Jadranka Loncarek ◽

Tim Stearns

Keyword(s):

Binding Protein ◽

Negative Regulator ◽

Microtubule Binding ◽

Centriole Duplication ◽

Mouse Double Minute ◽

Multiciliated Cells ◽

Dividing Cells ◽

Mouse Cells

Mouse double-minute 1 ( Mdm1) was originally identified as a gene amplified in transformed mouse cells and more recently as being highly up-regulated during differentiation of multiciliated epithelial cells, a specialized cell type having hundreds of centrioles and motile cilia. Here we show that the MDM1 protein localizes to centrioles of dividing cells and differentiating multiciliated cells. 3D-SIM microscopy showed that MDM1 is closely associated with the centriole barrel, likely residing in the centriole lumen. Overexpression of MDM1 suppressed centriole duplication, whereas depletion of MDM1 resulted in an increase in granular material that likely represents early intermediates in centriole formation. We show that MDM1 binds microtubules in vivo and in vitro. We identified a repeat motif in MDM1 that is required for efficient microtubule binding and found that these repeats are also present in CCSAP, another microtubule-binding protein. We propose that MDM1 is a negative regulator of centriole duplication and that its function is mediated through microtubule binding.

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