Interaction specificity between the chaperone and proteolytic components of the cyanobacterial Clp protease

2012 ◽  
Vol 446 (2) ◽  
pp. 311-320 ◽  
Author(s):  
Anders Tryggvesson ◽  
Frida M. Ståhlberg ◽  
Axel Mogk ◽  
Kornelius Zeth ◽  
Adrian K. Clarke
Keyword(s):  
Escherichia Coli ◽  
Active Sites ◽  
Terminal Sequence ◽  
Core Complex ◽  
Clp Protease ◽  
E Coli ◽  
Loop Region ◽  
N Terminus ◽  
Specific Association ◽  
The Stability

The Clp protease is conserved among eubacteria and most eukaryotes, and uses ATP to drive protein substrate unfolding and translocation into a chamber of sequestered proteolytic active sites. In plant chloroplasts and cyanobacteria, the essential constitutive Clp protease consists of the Hsp100/ClpC chaperone partnering a proteolytic core of catalytic ClpP and noncatalytic ClpR subunits. In the present study, we have examined putative determinants conferring the highly specific association between ClpC and the ClpP3/R core from the model cyanobacterium Synechococcus elongatus. Two conserved sequences in the N-terminus of ClpR (tyrosine and proline motifs) and one in the N-terminus of ClpP3 (MPIG motif) were identified as being crucial for the ClpC–ClpP3/R association. These N-terminal domains also influence the stability of the ClpP3/R core complex itself. A unique C-terminal sequence was also found in plant and cyanobacterial ClpC orthologues just downstream of the P-loop region previously shown in Escherichia coli to be important for Hsp100 association to ClpP. This R motif in Synechococcus ClpC confers specificity for the ClpP3/R core and prevents association with E. coli ClpP; its removal from ClpC reverses this core specificity.

scholarly journals In Vitro and In Vivo Assessment of the Potential of Escherichia coli Phages to Treat Infections and Survive Gastric Conditions

2021 ◽  
Vol 9 (9) ◽  
pp. 1869
Author(s):  
Joanna Kaczorowska ◽  
Eoghan Casey ◽  
Gabriele A. Lugli ◽  
Marco Ventura ◽  
David J. Clarke ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Galleria Mellonella ◽  
Enterotoxigenic Escherichia Coli ◽  
E Coli ◽  
Composite Mixture ◽  
Ph Conditions ◽  
The Stability ◽  
Higher Sensitivity

Enterotoxigenic Escherichia coli (ETEC) and Shigella ssp. infections are associated with high rates of mortality, especially in infants in developing countries. Due to increasing levels of global antibiotic resistance exhibited by many pathogenic organisms, alternative strategies to combat such infections are urgently required. In this study, we evaluated the stability of five coliphages (four Myoviridae and one Siphoviridae phage) over a range of pH conditions and in simulated gastric conditions. The Myoviridae phages were stable across the range of pH 2 to 7, while the Siphoviridae phage, JK16, exhibited higher sensitivity to low pH. A composite mixture of these five phages was tested in vivo in a Galleria mellonella model. The obtained data clearly shows potential in treating E. coli infections prophylactically.

scholarly journals Cloning of the Human Cytomegalovirus (HCMV) Genome as an Infectious Bacterial Artificial Chromosome in Escherichia coli: a New Approach for Construction of HCMV Mutants

1999 ◽  
Vol 73 (10) ◽  
pp. 8320-8329 ◽  
Author(s):  
Eva-Maria Borst ◽  
Gabriele Hahn ◽  
Ulrich H. Koszinowski ◽  
Martin Messerle
Keyword(s):  
Escherichia Coli ◽  
Human Cytomegalovirus ◽  
Human Fibroblasts ◽  
Artificial Chromosome ◽  
Bacterial Artificial Chromosomes ◽  
Reading Frame ◽  
E Coli ◽  
Artificial Chromosomes ◽  
Internal Repeat ◽  
The Stability

ABSTRACT We have recently introduced a novel procedure for the construction of herpesvirus mutants that is based on the cloning and mutagenesis of herpesvirus genomes as infectious bacterial artificial chromosomes (BACs) in Escherichia coli (M. Messerle, I. Crnković, W. Hammerschmidt, H. Ziegler, and U. H. Koszinowski, Proc. Natl. Acad. Sci. USA 94:14759–14763, 1997). Here we describe the application of this technique to the human cytomegalovirus (HCMV) strain AD169. Since it was not clear whether the terminal and internal repeat sequences of the HCMV genome would give rise to recombination, the stability of the cloned HCMV genome was examined during propagation inE. coli, during mutagenesis, and after transfection in permissive fibroblasts. Interestingly, the HCMV BACs were frozen in defined conformations in E. coli. The transfection of the HCMV BACs into human fibroblasts resulted in the reconstitution of infectious virus and isomerization of the reconstituted genomes. The power of the BAC mutagenesis procedure was exemplarily demonstrated by the disruption of the gpUL37 open reading frame. The transfection of the mutated BAC led to plaque formation, indicating that the gpUL37 gene product is dispensable for growth of HCMV in fibroblasts. The new procedure will considerably speed up the construction of HCMV mutants and facilitate genetic analysis of HCMV functions.

scholarly journals Association of ω with the C-Terminal Region of the β′ Subunit Is Essential for Assembly of RNA Polymerase in Mycobacterium tuberculosis

2018 ◽  
Vol 200 (12) ◽  
Author(s):  
Chunyou Mao ◽  
Yan Zhu ◽  
Pei Lu ◽  
Lipeng Feng ◽  
Shiyun Chen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Tuberculosis ◽  
Rna Polymerase ◽  
Essential Role ◽  
Β Subunit ◽  
Content Type ◽  
E Coli ◽  
Core Enzyme ◽  
Loop Region

ABSTRACT The ω subunit is the smallest subunit of bacterial RNA polymerase (RNAP). Although homologs of ω are essential in both eukaryotes and archaea, this subunit has been known to be dispensable for RNAP in Escherichia coli and in other bacteria. In this study, we characterized an indispensable role of the ω subunit in Mycobacterium tuberculosis . Unlike the well-studied E. coli RNAP, the M. tuberculosis RNAP core enzyme cannot be functionally assembled in the absence of the ω subunit. Importantly, substitution of M. tuberculosis ω with ω subunits from E. coli or Thermus thermophilus cannot restore the assembly of M. tuberculosis RNAP. Furthermore, by replacing different regions in M. tuberculosis ω with the corresponding regions from E. coli ω, we found a nonconserved loop region in M. tuberculosis ω essential for its function in RNAP assembly. From RNAP structures, we noticed that the location of the C-terminal region of the β′ subunit (β′CTD) in M. tuberculosis RNAP but not in E. coli or T. thermophilus RNAP is close to the ω loop region. Deletion of this β′CTD in M. tuberculosis RNAP destabilized the binding of M. tuberculosis ω on RNAP and compromised M. tuberculosis core assembly, suggesting that these two regions may function together to play a role in ω-dependent RNAP assembly in M. tuberculosis . Sequence alignment of the ω loop and the β′CTD regions suggests that the essential role of ω is probably restricted to mycobacteria. Together, our study characterized an essential role of M. tuberculosis ω and highlighted the importance of the ω loop region in M. tuberculosis RNAP assembly. IMPORTANCE DNA-dependent RNA polymerase (RNAP), which consists of a multisubunit core enzyme (α 2 ββ′ω) and a dissociable σ subunit, is the only enzyme in charge of transcription in bacteria. As the smallest subunit, the roles of ω remain the least well studied. In Escherichia coli and some other bacteria, the ω subunit is known to be nonessential for RNAP. In this study, we revealed an essential role of the ω subunit for RNAP assembly in the human pathogen Mycobacterium tuberculosis , and a mycobacterium-specific ω loop that plays a role in this function was also characterized. Our study provides fresh insights for further characterizing the roles of bacterial ω subunit.

scholarly journals Escherichia coli alkaline phosphatase. Kinetic studies with the tetrameric enzyme

1972 ◽  
Vol 126 (5) ◽  
pp. 1081-1090 ◽  
Author(s):  
S. E. Halford ◽  
M. J. Schlesinger ◽  
H. Gutfreund
Keyword(s):  
Escherichia Coli ◽  
Alkaline Phosphatase ◽  
Steady State ◽  
Analytical Ultracentrifugation ◽  
Kinetic Studies ◽  
Product Formation ◽  
E Coli ◽  
Enzyme Subunits ◽  
Self Association ◽  
The Stability

1. The stability of the tetrameric form of Escherichia coli alkaline phosphatase was examined by analytical ultracentrifugation. 2. The stopped-flow technique was used to study the hydrolysis of nitrophenyl phosphates by the alkaline phosphatase tetramer at pH7.5 and 8.3. In both cases transient product formation was observed before the steady state was attained. Both transients consisted of the liberation of 1mol of nitrophenol/2mol of enzyme subunits within the dead-time of the apparatus. The steady-state rates were identical with those observed with the dimer under the same conditions. 3. The binding of 2-hydroxy-5-nitrobenzyl phosphonate to the alkaline phosphatase tetramer was studied by the temperature-jump technique. The self-association of two dimers to form the tetramer is linked to a conformation change within the dimer. This accounts for the differences between the transient phases in the reactions of the dimer and the tetramer with substrate. 4. Addition of Pi to the alkaline phosphatase tetramer caused it to dissociate into dimers. The tetramer is unable to bind this ligand. It is suggested that the tetramer undergoes a compulsory dissociation before the completion of its first turnover with substrate. 5. On the basis of these findings a mechanism is proposed for the involvement of the alkaline phosphatase tetramer in the physiology of E. coli.

scholarly journals Effect of mutations in the transmethylase and dehydrogenase/chelatase domains of sirohaem synthase (CysG) on sirohaem and cobalamin biosynthesis

1998 ◽  
Vol 330 (1) ◽  
pp. 121-129 ◽  
Author(s):  
C. Sarah WOODCOCK ◽  
Evelyne RAUX ◽  
Florence LEVILLAYER ◽  
Claude THERMES ◽  
Alain RAMBACH ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Catalytic Activity ◽  
Catalytic Domain ◽  
Conserved Residues ◽  
Pseudomonas Denitrificans ◽  
E Coli ◽  
Cobalamin Biosynthesis ◽  
N Terminus ◽  
Low Levels ◽  
Cobyric Acid

The Escherichia coli CysG protein (sirohaem synthase) catalyses four separate reactions that are required for the transformation of uroporphyrinogen III into sirohaem, initially two S-adenosyl-L-methionine-dependent transmethylations at positions 2 and 7, mediated through the C-terminal, or CysGA, catalytic domain of the protein, and subsequently a ferrochelation and dehydrogenation, mediated through the N-terminal, or CysGB, catalytic domain of the enzyme. This report describes how the deletion of the NAD+-binding site of CysG, located within the first 35 residues of the N-terminus, is detrimental to the activity of CysGB but does not affect the catalytic activity of CysGA, whereas the mutation of a number of phylogenetically conserved residues within CysGA is detrimental to the transmethylation reaction but does not affect the activity of CysGB. Further studies have shown that CysGB is not essential for cobalamin biosynthesis because the presence of the Salmonella typhimurium CobI operon with either cysGA or the Pseudomonas denitrificans cobA are sufficient for the synthesis of cobyric acid in an E. coli cysG deletion strain. Evidence is also presented to suggest that a gene within the S. typhimurium CobI operon might act as a chelatase that, at low levels of cobalt, is able to aid in the synthesis of sirohaem.

In vivo associations of Escherichia coli NarJ with a peptide of the first 50 residues of nitrate reductase catalytic subunit NarG

2009 ◽  
Vol 55 (2) ◽  
pp. 179-188 ◽  
Author(s):  
Haiming Li ◽  
Raymond J. Turner
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Catalytic Subunit ◽  
Bimolecular Fluorescence Complementation ◽  
E Coli ◽  
N Terminus ◽  
Tat System

The catalytic subunit of many Escherichia coli redox enzymes bares a twin-arginine translocation (Tat)-dependent signal peptide in its precursor, which directs the redox enzyme complex to this Sec-independent pathway. NarG of the E. coli nitrate reductase NarGHI complex possesses a vestige twin-arginine motif at its N terminus. During the cofactor insertion, and assembly and folding of the NarG–NarH complex, a chaperone protein, NarJ, is thought to interact with the N terminus and an unknown second site of NarG. Our previous in vitro study provided evidence that NarJ’s role shows some Tat system dependence. In this work, we investigated the associations of NarJ with a peptide of the first 50 residues of NarG (NarG50) in living cells. Two approaches were used: the Förster resonance energy transfer (FRET) based on yellow fluorescent protein – cyan fluorescent protein (YFP–CFP) and the bimolecular fluorescence complementation (BiFC). Compared with the wild-type (WT) E. coli cotransformants expressing both NarJ–YFP and NarG50–CFP, tat gene mutants gave an apparent FRET efficiency (Eapp) that was on the order of 25%–40% lower. These experiments implied a Tat system dependency of the in vivo associations between NarJ and the NarG50 peptide. In the BiFC assay, a 4-fold lower specific fluorescence intensity was observed for the E. coli WT cotransformants expressing both NarJ–Yc and NarG50–Yn than for its tat mutants, again suggesting a Tat dependence of the interactions. Fluorescence microscopy showed a “dot”/unipolar distribution of the reassembled YFP–NarJ:NarG50 both in WT and tat mutants, demonstrating a distinct localization of the interaction. Thus, although the degree of the interaction shows Tat dependence, the cell localization is less so. Taken together, these data further support that NarJ’s activity on NarG may be assisted by the Tat system.

scholarly journals Different Processing of an mRNA Species inBacillus subtilis and Escherichia coli

2000 ◽  
Vol 182 (3) ◽  
pp. 689-695 ◽  
Author(s):  
Martin Persson ◽  
Elisabeth Glatz ◽  
Blanka Rutberg
Keyword(s):  
Escherichia Coli ◽  
Steady State ◽  
Inverted Repeat ◽  
Fusion Transcript ◽  
Temperature Sensitive ◽  
Rnase Iii ◽  
Transcription Terminator ◽  
E Coli ◽  
Steady State Levels ◽  
The Stability

ABSTRACT Expression of the Bacillus subtilis glpD gene, which encodes glycerol-3-phosphate (G3P) dehydrogenase, is controlled by termination or antitermination of transcription. The untranslated leader sequence of glpD contains an inverted repeat that gives rise to a transcription terminator. In the presence of G3P, the antiterminator protein GlpP binds toglpD leader mRNA and promotes readthrough of the terminator. Certain mutations in the inverted repeat of theglpD leader result in GlpP-independent, temperature-sensitive (TS) expression of glpD. The TS phenotype is due to temperature-dependent degradation of theglpD mRNA. In the presence of GlpP, theglpD mRNA is stabilized. glpDleader-lacZ fusions were integrated into the chromosomes ofB. subtilis and Escherichia coli. Determination of steady-state levels of fusion mRNA in B. subtilis showed that the stability of the fusion mRNA is determined by theglpD leader part. Comparison of steady-state levels and half-lives of glpD leader-lacZ fusion mRNA inB. subtilis and E. coli revealed significant differences. A glpD leader-lacZ fusion transcript that was unstable in B. subtilis was considerably more stable in E. coli. GlpP, which stabilizes the transcript in B. subtilis, did not affect its stability in E. coli. Primer extension analysis showed that theglpD leader-lacZ fusion transcript is processed differently in B. subtilis and in E. coli. The dominating cleavage site in E. coli was barely detectable in B. subtilis. This site was shown to be a target ofE. coli RNase III.

scholarly journals Escherichia coliflagellar serotyping is as reliable as it has always been!

1987 ◽  
Vol 98 (2) ◽  
pp. 221-222 ◽  
Author(s):  
Frits Ørskov ◽  
Ida Ørskov ◽  
K. A. Bettelheim
Keyword(s):  
Escherichia Coli ◽  
Dna Analysis ◽  
Epidemiological Evidence ◽  
Single Strain ◽  
E Coli ◽  
Electrophoretic Patterns ◽  
Cast Doubt ◽  
The Stability ◽  
H Serotypes

In a recent paper in this Journal by Marshallet al.(1985), which described the bacterial endonuclease DNA analysis (BRENDA) of severalEscherichia coli0126 strains, the following statement was found in the summary: ‘The isolates from the outbreak produced indistinguishable DNA electrophoretic patterns in spite of their assignment to seven different H serotypes… These results support the epidemiological evidence that a single-strain outbreak had occurred, and they cast doubt on the value of H typing for this particular investigation.’ The same 0126 strains that were enterotoxigenic (ST) were treated in two earlier papers (Bettelheim & Reeve, 1982; Bettelheim, 1984) and also on these two earlier occasions the authors raised doubt about the stability ofE. coliH typing results.

scholarly journals Regulation of RraA, a Protein Inhibitor of RNase E-Mediated RNA Decay

2006 ◽  
Vol 188 (9) ◽  
pp. 3257-3263 ◽  
Author(s):  
Meng Zhao ◽  
Li Zhou ◽  
Yasuaki Kawarasaki ◽  
George Georgiou
Keyword(s):  
Escherichia Coli ◽  
Intergenic Region ◽  
Ectopic Expression ◽  
Dependent Manner ◽  
Feedback Circuit ◽  
Rnase E ◽  
Protein Inhibitor ◽  
E Coli ◽  
The Stability ◽  
Fusion Analysis

ABSTRACT The recently discovered RraA protein acts as an inhibitor of the essential endoribonuclease RNase E, and we demonstrated that ectopic expression of RraA affects the abundance of more than 700 transcripts in Escherichia coli (K. Lee, X. Zhan, J. Gao, J. Qiu, Y. Feng, R. Meganathan, S. N. Cohen, and G. Georgiou, Cell 114:623-634, 2003). We show that rraA is expressed from its own promoter, P rraA , located in the menA-rraA intergenic region. Primer extension and lacZ fusion analysis revealed that transcription from P rraA is elevated upon entry into stationary phase in a σs-dependent manner. In addition, the stability of the rraA transcript is dependent on RNase E activity, suggesting the involvement of a feedback circuit in the regulation of the RraA level in E. coli.

The ATPase complex of Escherichia coli

1984 ◽  
Vol 62 (11) ◽  
pp. 1190-1197 ◽  
Author(s):  
Philip D. Bragg
Keyword(s):  
Escherichia Coli ◽  
Chemical Modification ◽  
Active Site ◽  
Active Sites ◽  
Transmembrane Protein ◽  
Proton Translocation ◽  
Amino Terminal ◽  
Loop Region ◽  
Cytoplasmic Surface ◽  
Hydrolysis Of

The ATPase (ATP synthase) complex of Escherichia coli is composed of an extrinsic membrane protein (ECF1), which contains the active site for ATP formation and hydrolysis, and is attached to ECF0, a transmembrane protein through which protons move to or from the active site on ECF1. ECF1 is composed of five subunits (α–ε) with a stoichiometry of α3β3γδε. The stoichiometry of the three subunits (a–c) of ECF0 is probably a1b2c10–15. In addition to 3 mol tightly bound adenine nucleotide/mol ECF1, three other "exchangeable" nucleotide binding sites can be detected. These sites are still present in the α and β subunit defective ECF1 of uncA401 and uncD412 mutants, although some changes in the tightness of binding are evident. The active sites of ECF1 require normal a and p subunits and may be present at αβ subunit interfaces. Hydrolysis of ATP requires cooperative interactions between α and β subunits. At low concentrations of ATP, in the absence of added divalent cations, hydrolysis of this substrate can occur at a single site without release of the product. This is consistent with alternating or sequential site mechanisms for ATP hydrolysis or synthesis. Predictions of secondary and tertiary structures from the known primary amino acid sequences of polypeptides a, b, and c have led to the following conclusions. Polypeptide a forms six or seven transmembrane a helices. The amino-terminal sequence of polypeptide b spans the membrane, but most of the protein is exposed on the cytoplasmic surface of the membrane where it can be cleaved by proteases in vitro. Polypeptide c consists of two nonpolar membrane-spanning α helices linked by a polar segment at the cytoplasmic surface of the membrane. This loop region interacts with ECF1 or is close to the ECF1-binding site. This is shown by competition between ECF1 and antibody for binding to polypeptide c. Chemical modification of arginyl residues in the loop region of polypeptide c inhibits ECF1 binding. Protease cleavage of polypeptide b affects, but does not abolish, binding of ECF1 to ECF0. Presumably, polypeptide b interacts with ECF1 also. The individual roles of the ECF0 polypeptides in proton translocation are not clear. Mutants in any of the three polypeptides may be defective in proton translocation. However, mutant and chemical modification studies support a role for the polypeptide c oligomer in the transmembrane proton pathway.

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