scholarly journals PepN is the major aminopeptidase in Escherichia coli: insights on substrate specificity and role during sodium-salicylate-induced stress

Microbiology ◽  
2003 ◽  
Vol 149 (12) ◽  
pp. 3437-3447 ◽  
Author(s):  
Dilip Chandu ◽  
Dipankar Nandi
Keyword(s):  
Escherichia Coli ◽  
Sodium Salicylate ◽  
Physiological Role ◽  
Negative Regulator ◽  
Aminopeptidase Activity ◽  
Peptidase Activity ◽  
E Coli ◽  
Induced Stress ◽  
Hydrolysis Of

PepN and its homologues are involved in the ATP-independent steps (downstream processing) during cytosolic protein degradation. To obtain insights into the contribution of PepN to the peptidase activity in Escherichia coli, the hydrolysis of a selection of endopeptidase and exopeptidase substrates was studied in extracts of wild-type strains and two pepN mutants, 9218 and DH5αΔpepN. Hydrolysis of three of the seven endopeptidase substrates tested was reduced in both pepN mutants. Similar studies revealed that hydrolysis of 10 of 14 exopeptidase substrates studied was greatly reduced in both pepN mutants. This decreased ability to cleave these substrates is pepN-specific as there is no reduction in the ability to hydrolyse exopeptidase substrates in E. coli mutants lacking other peptidases, pepA, pepB or pepE. PepN overexpression complemented the hydrolysis of the affected exopeptidase substrates. These results suggest that PepN is responsible for the majority of aminopeptidase activity in E. coli. Further in vitro studies with purified PepN revealed a preference to cleave basic and small amino acids as aminopeptidase substrates. Kinetic characterization revealed the aminopeptidase cleavage preference of E. coli PepN to be Arg>Ala>Lys>Gly. Finally, it was shown that PepN is a negative regulator of the sodium-salicylate-induced stress in E. coli, demonstrating a physiological role for this aminoendopeptidase under some stress conditions.

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

Pathogens ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 268 ◽  
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.

Use of [1-14C]oleate labelled autoclaved Escherichia coli as a membranous substrate for measurement of in vitro phospholipase D activity

1992 ◽  
Vol 70 (1) ◽  
pp. 43-48 ◽  
Author(s):  
S. S. Ghosh ◽  
Richard C. Franson
Keyword(s):  
Escherichia Coli ◽  
Phosphatidic Acid ◽  
Phospholipase D ◽  
Phospholipase A ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
E Coli ◽  
Streptomyces Chromofuscus ◽  
Hydrolysis Of

Autoclaved Escherichia coli labelled with [1-14C]oleate in the 2-acyl position have been used extensively to measure phospholipase A2 activity in vitro. The present study demonstrates that this membranous substrate is also useful for the measurement of in vitro phospholipase D activity. Phospholipase D from Streptomyces chromofuscus catalyzed the hydrolysis of [1-14C]oleate labelled, autoclaved E. coli optimally at pH 7.0–8.0 to generate [14C]phosphatidic acid in the presence of 5 mM added Ca2+. Other divalent cations would not substitute for Ca2+. Activity was linear with time and protein up to 30% of the hydrolysis of substrate. Phospholipase D activity was stimulated in a dose-dependent manner by the addition of Triton X-100. The activity was increased 5.5-fold with 0.05% Triton, a concentration that totally inhibited hydrolysis of E. coli by human synovial fluid phospholipase A2. Accumulation of [14C]diglyceride was observed after 10 min of incubation. This accumulation was inhibited by NaF (IC50 = 18 μM) or propanolol (IC50 = 180 μM) suggesting the S. chromofuscus phospholipase D was contaminated with phosphatidate phosphohydrolase. Phosphatidic acid released by the action of cabbage phospholipase D was converted to phosphatidylethanol in an ethanol concentration dependent manner. These results demonstrate that [1-14C]oleate labelled, autoclaved E. coli can be used to measure phospholipase D activity by monitoring accumulation of either [14C]phosphatidic acid or [14C]phosphatidylethanol.Key words: Escherichia coli, substrate, phospholipase D, Streptomyces chromofuscus, sodium fluoride, propranolol.

scholarly journals Alteration of the Repressor Activity of MarR, the Negative Regulator of the Escherichia coli marRAB Locus, by Multiple Chemicals In Vitro

1999 ◽  
Vol 181 (15) ◽  
pp. 4669-4672 ◽  
Author(s):  
Michael N. Alekshun ◽  
Stuart B. Levy
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Sodium Salicylate ◽  
Negative Regulator ◽  
Multiple Antibiotic Resistance ◽  
Whole Cells ◽  
Repressor Activity

ABSTRACT MarR negatively regulates expression of the multiple antibiotic resistance operon (marRAB) in Escherichia coli. In this study, it was demonstrated that sodium salicylate, plumbagin, 2,4-dinitrophenol, and menadione–inducers of the marRABoperon in whole cells–all interfered with the repressor activity of MarR in vitro. It is proposed that these compounds can interact directly with MarR to affect its repressor activity.

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

2020 ◽  
Vol 88 (6) ◽  
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.

scholarly journals The Crystal Structure of the Zinc Phosphodiesterase from Escherichia coli Provides Insight into Function and Cooperativity of tRNase Z-Family Proteins

2006 ◽  
Vol 188 (4) ◽  
pp. 1607-1614 ◽  
Author(s):  
Brenda Kostelecky ◽  
Ehmke Pohl ◽  
Andreas Vogel ◽  
Oliver Schilling ◽  
Wolfram Meyer-Klaucke
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Metal Binding ◽  
Thermotoga Maritima ◽  
Physiological Role ◽  
E Coli ◽  
Ability To Act ◽  
Trnase Z ◽  
Insight Into

ABSTRACT The elaC gene product from Escherichia coli, ZiPD, is a 3′ tRNA-processing endonuclease belonging to the tRNase Z family of enzymes that have been identified in a wide variety of organisms. In contrast to the elaC homologue from Bacillus subtilis, E. coli elaC is not essential for viability, and although both enzymes process only precursor tRNA (pre-tRNA) lacking a CCA triplet at the 3′ end in vitro, the physiological role of ZiPD remains enigmatic because all pre-tRNA species in E. coli are transcribed with the CCA triplet. We present the first crystal structure of ZiPD determined by multiple anomalous diffraction at a resolution of 2.9 Å. This structure shares many features with the tRNase Z enzymes from B. subtilis and Thermotoga maritima, but there are distinct differences in metal binding and overall domain organization. Unlike the previously described homologous structures, ZiPD dimers display crystallographic symmetry and fully loaded metal sites. The ZiPD exosite is similar to that of the B. subtilis enzyme structurally, but its position with respect to the protein core differs substantially, illustrating its ability to act as a clamp in binding tRNA. Furthermore, the ZiPD crystal structure presented here provides insight into the enzyme's cooperativity and assists the ongoing attempt to elucidate the physiological function of this protein.

scholarly journals A Truncated Soluble Bacillus Signal Peptidase Produced in Escherichia coli Is Subject to Self-Cleavage at Its Active Site

2000 ◽  
Vol 182 (20) ◽  
pp. 5765-5770 ◽  
Author(s):  
M. L. van Roosmalen ◽  
J. D. H. Jongbloed ◽  
A. Kuipers ◽  
G. Venema ◽  
S. Bron ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Signal Peptidase ◽  
Soluble Form ◽  
Peptidase Activity ◽  
Mutant Proteins ◽  
E Coli ◽  
Amino Terminal ◽  
Complete Inactivation ◽  
High Level

ABSTRACT Soluble forms of Bacillus signal peptidases which lack their unique amino-terminal membrane anchor are prone to degradation, which precludes their high-level production in the cytoplasm ofEscherichia coli. Here, we show that the degradation of soluble forms of the Bacillus signal peptidase SipS is largely due to self-cleavage. First, catalytically inactive soluble forms of this signal peptidase were not prone to degradation; in fact, these mutant proteins were produced at very high levels in E. coli. Second, the purified active soluble form of SipS displayed self-cleavage in vitro. Third, as determined by N-terminal sequencing, at least one of the sites of self-cleavage (between Ser15 and Met16 of the truncated enzyme) strongly resembles a typical signal peptidase cleavage site. Self-cleavage at the latter position results in complete inactivation of the enzyme, as Ser15 forms a catalytic dyad with Lys55. Ironically, self-cleavage between Ser15 and Met16 cannot be prevented by mutagenesis of Gly13 and Ser15, which conform to the −1, −3 rule for signal peptidase recognition, because these residues are critical for signal peptidase activity.

scholarly journals Characterization of the Autocleavage Process of the Escherichia coli HtrA Protein: Implications for its Physiological Role

2008 ◽  
Vol 191 (6) ◽  
pp. 1924-1932 ◽  
Author(s):  
Ahmad Jomaa ◽  
Jack Iwanczyk ◽  
Julie Tran ◽  
Joaquin Ortega
Keyword(s):  
Escherichia Coli ◽  
Physiological Role ◽  
Protein S ◽  
Stress Conditions ◽  
Cage Structure ◽  
Protein Substrates ◽  
Cleavage Process ◽  
Hydrolysis Of

ABSTRACT The Escherichia coli HtrA protein is a periplasmic protease/chaperone that is upregulated under stress conditions. The protease and chaperone activities of HtrA eliminate or refold damaged and unfolded proteins in the bacterial periplasm that are generated upon stress conditions. In the absence of substrates, HtrA oligomerizes into a hexameric cage, but binding of misfolded proteins transforms the hexamers into bigger 12-mer and 24-mer cages that encapsulate the substrates for degradation or refolding. HtrA also undergoes partial degradation as a consequence of self-cleavage of the mature protein, producing short-HtrA protein (s-HtrA). The aim of this study was to examine the physiological role of this self-cleavage process. We found that the only requirement for self-cleavage of HtrA into s-HtrA in vitro was the hydrolysis of protein substrates. In fact, peptides resulting from the hydrolysis of the protein substrates were sufficient to induce autocleavage. However, the continuous presence of full-length substrate delayed the process. In addition, we observed that the hexameric cage structure is required for autocleavage and that s-HtrA accumulates only late in the degradation reaction. These results suggest that self-cleavage occurs when HtrA reassembles back into the resting hexameric structure and peptides resulting from substrate hydrolysis are allosterically stimulating the HtrA proteolytic activity. Our data support a model in which the physiological role of the self-cleavage process is to eliminate the excess of HtrA once the stress conditions cease.

scholarly journals Probing the active site of YjeE: a vital Escherichia coli protein of unknown function

2004 ◽  
Vol 384 (3) ◽  
pp. 577-584 ◽  
Author(s):  
Abdellah ALLALI-HASSANI ◽  
Tracey L. CAMPBELL ◽  
Andy HO ◽  
Jeffrey W. SCHERTZER ◽  
Eric D. BROWN
Keyword(s):  
Escherichia Coli ◽  
Unknown Function ◽  
Equilibrium Dialysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Wild Type ◽  
E Coli ◽  
High Affinity ◽  
Hydrolysis Of

In the study described here, we have taken steps to characterize the YjeE protein, an Escherichia coli protein of unknown function that is essential for bacterial viability. YjeE represents a protein family whose members are broadly conserved in bacteria, absent from eukaryotes and contain both Walker A and B motifs, characteristic of P-loop ATPases. We have revisited the dispensability of the yjeE gene in E. coli and describe efforts to probe the function of the YjeE protein with in vitro biochemistry. We have looked critically for ATPase activity in the recombinant E. coli protein and have made vigilant use of site-directed variants in the Walker A [K41A (Lys41→Ala) and T42A] and putative Walker B (D80Q) motifs. We noted that any hydrolysis of ATP by the wild-type E. coli protein might be attributed to background ATPase, since it was not appreciably different from that of the variants. To overcome potential contaminants, we turned to crystalline pure YjeE protein from Haemophilus influenzae that was found to hydrolyse ATP at a slow rate (kcat=1 h−1). We have also shown high-affinity binding to YjeE by ADP using equilibrium dialysis (Kd=32 μM) and by fluorescence resonance energy transfer from a conserved tryptophan in YjeE to a fluorescent derivative of ADP, 2′-/3′-O-(N-methylanthraniloyl)adenosine 5′-O-diphosphate (Kd=8 μM). Walker motif variants were notably impaired for ADP binding and T42A and D80Q mutations in yjeE were incapable of complementing the yjeE deletion strain.

scholarly journals Biophysical characterization of two commercially available preparations of the drug containing Escherichia coli L-Asparaginase 2

2020 ◽  
Author(s):  
Talita Stelling de Araújo ◽  
Sandra M. N. Scapin ◽  
William de Andrade ◽  
Maira Fasciotti ◽  
Mariana T. Q. de Magalhães ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Lymphoblastic Leukemia ◽  
Biophysical Characterization ◽  
E Coli ◽  
Terminal Loop ◽  
Hydrolysis Of

AbstractThe hydrolysis of asparagine and glutamine by L-asparaginase has been used to treat acute lymphoblastic leukemia for over four decades. Each L-asparaginase monomer has a long loop that closes over the active site upon substrate binding, acting as a lid. Here we present a comparative study two commercially available preparations of the drug containing Escherichia coli L-Asparaginase 2, performed by a comprehensive array of biophysical and biochemical approaches. We report the oligomeric landscape and conformational and dynamic plasticity of E. coli type 2 L-asparaginase (EcA2) present in two different formulations, and its relationship with L-aspartic acid, which is present in Aginasa, but not in Leuginase. EcA2 shows a composition of monomers and oligomers up to tetramers, which is mostly not altered in the presence of L-Asp. The N-terminal loop of Leuginase, which is part of the active site is flexibly disordered, but gets ordered as in Aginasa in the presence os L-Asp, while L-Glu only does so to a limited extent. Ion-mobility spectrometry–mass spectrometry reveals two conformers for the monomeric EcA2, one of which can selectively bind to L-Asp and L-Glu. Aginasa has higher resistance to in vitro proteolysis than Leuginase, and this is directly related to the presence of L-Asp.

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

2009 ◽  
Vol 192 (1) ◽  
pp. 242-255 ◽  
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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