Regulation of RcsA by the ClpYQ (HslUV) protease in Escherichia coli

Microbiology ◽  
2004 ◽  
Vol 150 (2) ◽  
pp. 437-446 ◽  
Author(s):  
Mei-Shiue Kuo ◽  
Kuei-Peng Chen ◽  
Whi Fin Wu
Keyword(s):  
Escherichia Coli ◽  
Lon Protease ◽  
Specific Substrate ◽  
Galactosidase Activity ◽  
Wild Type ◽  
Reporter Construct ◽  
Fusion Construct ◽  
In The Wild ◽  
A Cell ◽  
Cell Division Inhibitor

Escherichia coli ClpYQ protease and Lon protease possess a redundant function for degradation of SulA, a cell division inhibitor. An experimental cue implied that the capsule synthesis activator RcsA, a known substrate of Lon, is probably a specific substrate for the ClpYQ protease. This paper shows that overexpression of ClpQ and ClpY suppresses the mucoid phenotype of a lon mutant. Since the cpsB (wcaB) gene, involved in capsule synthesis, is activated by RcsA, the reporter construct cpsB–lacZ was used to assay for β-galactosidase activity and thus follow RcsA stability. The expression of cpsB–lacZ was increased in double mutants of lon in combination with clpQ or/and clpY mutation(s) compared with the wild-type or lon single mutants. Overproduction of ClpYQ or ClpQ decreased cpsB–lacZ expression. Additionally, a PBAD–rcsA fusion construct showed quantitatively that an inducible RcsA activates cpsB–lacZ expression. The effect of RcsA on cpsB–lacZ expression was shown to be influenced by the ClpYQ activities. Moreover, a rcsA Red –lacZ translational fusion construct showed higher activity of RcsARed–LacZ in a clpQ clpY strain than in the wild-type. By contrast, overproduction of cellular ClpYQ resulted in decreased β-galactosidase levels of RcsARed–LacZ. Taken together, the data indicate that ClpYQ acts as a secondary protease in degrading the Lon substrate RcsA.

scholarly journals Mutants of Citrobacter freundii That Transport and Utilize Melibiose

1998 ◽  
Vol 180 (13) ◽  
pp. 3480-3482
Author(s):  
Noriko Okazaki ◽  
Xing Jue Xu ◽  
Toshi Shimamoto ◽  
Masayuki Kuroda ◽  
Thomas H. Wilson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Citrobacter Freundii ◽  
Sodium Ions ◽  
Galactosidase Activity ◽  
Transport Activity ◽  
Wild Type ◽  
Chromosomal Dna ◽  
In The Wild ◽  
Mutant Cells

ABSTRACT We have isolated mutants of Citrobacter freundii that can grow on melibiose. Inducible α-galactosidase activity and melibiose transport activity were detected in the mutant cells but not in the wild-type cells. We detected a DNA region which hybridized withmelB (the gene for the melibiose transporter) DNA ofEscherichia coli in the chromosomal DNA of wild-typeC. freundii. Protons, but not sodium ions, were found to be the coupling cations for melibiose (and methyl-β-d-thiogalactoside) transport in the mutant cells.

scholarly journals Escherichia coli MutM Suppresses Illegitimate Recombination Induced by Oxidative Stress

Genetics ◽  
1999 ◽  
Vol 151 (2) ◽  
pp. 439-446 ◽  
Author(s):  
Masaaki Onda ◽  
Katsuhiro Hanada ◽  
Hirokazu Kawachi ◽  
Hideo Ikeda
Keyword(s):  
Oxidative Stress ◽  
Escherichia Coli ◽  
Hydrogen Peroxide ◽  
Dna Damage ◽  
Double Strand Breaks ◽  
Illegitimate Recombination ◽  
Recombination Analysis ◽  
Wild Type ◽  
Strand Breaks ◽  
In The Wild

Abstract DNA damage by oxidative stress is one of the causes of mutagenesis. However, whether or not DNA damage induces illegitimate recombination has not been determined. To study the effect of oxidative stress on illegitimate recombination, we examined the frequency of λbio transducing phage in the presence of hydrogen peroxide and found that this reagent enhances illegitimate recombination. To clarify the types of illegitimate recombination, we examined the effect of mutations in mutM and related genes on the process. The frequency of λbio transducing phage was 5- to 12-fold higher in the mutM mutant than in the wild type, while the frequency in the mutY and mutT mutants was comparable to that of the wild type. Because 7,8-dihydro-8-oxoguanine (8-oxoG) and formamido pyrimidine (Fapy) lesions can be removed from DNA by MutM protein, these lesions are thought to induce illegitimate recombination. Analysis of recombination junctions showed that the recombination at Hotspot I accounts for 22 or 4% of total λbio transducing phages in the wild type or in the mutM mutant, respectively. The preferential increase of recombination at nonhotspot sites with hydrogen peroxide in the mutM mutant was discussed on the basis of a new model, in which 8-oxoG and/or Fapy residues may introduce double-strand breaks into DNA.

O6-methylguanine-DNA methyltransferase in wild-type and ada mutants of Escherichia coli

1982 ◽  
Vol 152 (1) ◽  
pp. 534-537
Author(s):  
S Mitra ◽  
B C Pal ◽  
R S Foote
Keyword(s):  
Escherichia Coli ◽  
Dna Methyltransferase ◽  
Wild Type ◽  
E Coli ◽  
Methylguanine Dna Methyltransferase ◽  
Synthetic Dna ◽  
In The Wild ◽  
Low Levels ◽  
Enzyme Molecules ◽  
Dna Substrate

O(6)-Methylguanine-DNA methyltransferase is induced in Escherichia coli during growth in low levels of N-methyl-N'-nitro-N-nitrosoguanidine. We have developed a sensitive assay for quantitating low levels of this activity with a synthetic DNA substrate containing 3H-labeled O(6)-methylguanine as the only modified base. Although both wild-type and adaptation-deficient (ada) mutants of E. coli contained low but comparable numbers (from 13 to 60) of the enzyme molecules per cell, adaptation treatment caused a significant increase of the enzyme in the wild type but not in the ada mutants, suggesting that the ada mutation is in a regulatory locus and not in the structural gene for the methyltransferase.

Sensitivity of normal and mutant strains of Escherichia coli to actinomycin-D

1972 ◽  
Vol 18 (6) ◽  
pp. 909-915 ◽  
Author(s):  
A. P. Singh ◽  
K.-J. Cheng ◽  
J. W. Costerton ◽  
E. S. Idziak ◽  
J. M. Ingram
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Wild Type Strain ◽  
Actinomycin D ◽  
Cytoplasmic Membrane ◽  
Cell Envelope ◽  
Coli Strain ◽  
Wild Type ◽  
Mutant Strains ◽  
In The Wild

The site of the cell barrier to actinomycin-D uptake was studied using a wild-type Escherichia coli strain P and its cell envelope-defective filamentous mutants, strains 6γ and 12γ, both of which 'leak' β-galactosidase and alkaline phosphatase into the medium during growth indicating both membrane and cell-wall defects. Actinomycin-D entered the cells of these two mutant strains as evidenced by the inhibition of both 14C-uracil incorporation and synthesis of the induced β-galactosidase system. Under similar conditions, no inhibition occurred in the wild-type strain and its sucrose-lysozyme prepared spheroplasts. Actinomycin-D did, however, inhibit the above-mentioned systems in the wild-type sucrose-lysozyme spheroplasts prepared in the presence of 2 mM EDTA. The experimental data indicate that although the cell wall may act as a primary barrier or sieve to actinomycin-D, the cytoplasmic membrane should be considered the final and determinative barrier to this antibiotic.

scholarly journals Plasmid DNA Production in Proteome-Reduced Escherichia coli

2020 ◽  
Vol 8 (9) ◽  
pp. 1444
Author(s):  
Mitzi de la Cruz ◽  
Elisa A. Ramírez ◽  
Juan-Carlos Sigala ◽  
José Utrilla ◽  
Alvaro R. Lara
Keyword(s):  
Escherichia Coli ◽  
Plasmid Dna ◽  
Type Strain ◽  
Wild Type Strain ◽  
The Novel ◽  
Wild Type ◽  
Batch Mode ◽  
Cell Factories ◽  
Starting Point ◽  
In The Wild

The design of optimal cell factories requires engineering resource allocation for maximizing product synthesis. A recently developed method to maximize the saving in cell resources released 0.5% of the proteome of Escherichia coli by deleting only three transcription factors. We assessed the capacity for plasmid DNA (pDNA) production in the proteome-reduced strain in a mineral medium, lysogeny, and terrific broths. In all three cases, the pDNA yield from biomass was between 33 and 53% higher in the proteome-reduced than in its wild type strain. When cultured in fed-batch mode in shake-flask, the proteome-reduced strain produced 74.8 mg L−1 pDNA, which was four times greater than its wild-type strain. Nevertheless, the pDNA supercoiled fraction was less than 60% in all cases. Deletion of recA increased the pDNA yields in the wild type, but not in the proteome-reduced strain. Furthermore, recA mutants produced a higher fraction of supercoiled pDNA, compared to their parents. These results show that the novel proteome reduction approach is a promising starting point for the design of improved pDNA production hosts.

scholarly journals Heteromeric Interactions among Nucleoid-Associated Bacterial Proteins: Localization of StpA-Stabilizing Regions in H-NS of Escherichia coli

2001 ◽  
Vol 183 (7) ◽  
pp. 2343-2347 ◽  
Author(s):  
Jörgen Johansson ◽  
Sven Eriksson ◽  
Berit Sondén ◽  
Sun Nyunt Wai ◽  
Bernt Eric Uhlin
Keyword(s):  
Escherichia Coli ◽  
Stable Form ◽  
Lon Protease ◽  
Wild Type ◽  
Bacterial Proteins ◽  
Regulatory Systems ◽  
Associated Proteins ◽  
Gene Regulatory

ABSTRACT The nucleoid-associated proteins H-NS and StpA inEscherichia coli bind DNA as oligomers and are implicated in gene regulatory systems. There is evidence for both homomeric and heteromeric H-NS–StpA complexes. The two proteins show differential turnover, and StpA was previously found to be subject to protease-mediated degradation by the Lon protease. We investigated which regions of the H-NS protein are able to prevent degradation of StpA. A set of truncated H-NS derivatives was tested for their ability to mediate StpA stability and to form heteromers in vitro. The data indicate that H-NS interacts with StpA at two regions and that the presence of at least one of the H-NS regions is necessary for StpA stability. Our results also suggest that a proteolytically stable form of StpA, StpAF21C, forms dimers, whereas wild-type StpA in the absence of H-NS predominantly forms tetramers or oligomers, which are more susceptible to proteolysis.

Regulation of SulA cleavage by Lon protease by the C-terminal amino acid of SulA, histidine

2001 ◽  
Vol 358 (2) ◽  
pp. 473-480 ◽  
Author(s):  
Yoshiyuki ISHII ◽  
Fumio AMANO
Keyword(s):  
Amino Acid ◽  
Protein A ◽  
Lon Protease ◽  
Histidine Residue ◽  
High Accumulation ◽  
C Terminus ◽  
A Cell ◽  
Cell Division Inhibitor

SulA protein, a cell division inhibitor in Escherichia coli, is degraded by Lon protease. The C-terminal eight residues of SulA have been shown to be recognized by Lon; however, it remains to be elucidated which amino acid in the C-terminus of SulA is critical for the recognition of SulA by Lon. To clarify this point, we constructed mutants of SulA with changes in the C-terminal residues, and examined the accumulation and stability of the resulting mutant SulA proteins in vivo. Substitution of the extreme C-terminal histidine residue with another amino acid led to marked accumulation and high stability of SulA in lon+ cells. A SulA mutant in which the C-terminal eight residues were deleted (SulAC161) showed high accumulation and stability, but the addition of histidine to the C-terminus of SulAC161 (SulAC161+H) made it labile. Similarly, SulAC161+H fused to maltose-binding protein (MBP–SulAC161+H) formed a tight complex with and was degraded rapidly by Lon in vitro. Histidine competitively inhibited the degradation of MBP–SulA by Lon, while other amino acids did not. These results suggest that the histidine residue at the extreme C-terminus of SulA is recognized specifically by Lon, leading to a high-affinity interaction between SulA and Lon.

scholarly journals Cephem Potentiation by Inactivation of Nonessential Genes Involved in Cell Wall Biogenesis of β-Lactamase-Producing Escherichia coli

2016 ◽  
Vol 61 (3) ◽  
Author(s):  
Kristin R. Baker ◽  
Helga Høeg Sigurðardóttir ◽  
Bimal Jana ◽  
Luca Guardabassi
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Lag Phase ◽  
Growth Curve Analysis ◽  
Wild Type ◽  
Content Type ◽  
Cell Wall Biogenesis ◽  
Genes Encoding ◽  
In The Wild ◽  
Nonessential Genes

ABSTRACT Reversal of antimicrobial resistance is an appealing and largely unexplored strategy in drug discovery. The objective of this study was to identify potential targets for “helper” drugs reversing cephem resistance in Escherichia coli strains producing β-lactamases. A CMY-2-encoding plasmid was transferred by conjugation to seven isogenic deletion mutants exhibiting cephem hypersusceptibility. The effect of each mutation was evaluated by comparing the MICs in the wild type and the mutant harboring the same plasmid. Mutation of two genes encoding proteins involved in cell wall biosynthesis, dapF and mrcB, restored susceptibility to cefoxitin (FOX) and reduced the MICs of cefotaxime and ceftazidime, respectively, from the resistant to the intermediate category according to clinical breakpoints. The same mutants harboring a CTX-M-1-encoding plasmid fell into the intermediate or susceptible category for all three drugs. Individual deletion of dapF and mrcB in a clinical isolate of CTX-M-15-producing E. coli sequence type 131 (ST131) resulted in partial reversal of ceftazidime and cefepime resistance but did not reduce MICs below susceptibility breakpoints. Growth curve analysis indicated no fitness cost in a ΔmrcB mutant, whereas a ΔdapF mutant had a 3-fold longer lag phase than the wild type, suggesting that drugs targeting DapF may display antimicrobial activity, in addition to synergizing with selected cephems. DapF appeared to be a potential FOX helper drug target candidate, since dapF inactivation resulted in synergistic potentiation of FOX in the genetic backgrounds tested. The study showed that individual inactivation of two nonessential genes involved in cell wall biogenesis potentiates cephem activity according to drug- and strain-specific patterns.

scholarly journals Exploring by Pulsed EPR the Electronic Structure of Ubisemiquinone Bound at the QH Site of Cytochrome bo3 from Escherichia coli with in Vivo13C-Labeled Methyl and Methoxy Substituents

2011 ◽  
Vol 286 (12) ◽  
pp. 10105-10114 ◽  
Author(s):  
Myat T. Lin ◽  
Alexander A. Shubin ◽  
Rimma I. Samoilova ◽  
Kuppala V. Narasimhulu ◽  
Amgalanbaatar Baldansuren ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Wild Type ◽  
Mutant Proteins ◽  
Pulsed Epr ◽  
Cytochrome Bo3 ◽  
Methoxy Groups ◽  
Unpaired Spin ◽  
In The Wild ◽  
Electron Reduction ◽  
The One

The cytochrome bo3 ubiquinol oxidase from Escherichia coli resides in the bacterial cytoplasmic membrane and catalyzes the two-electron oxidation of ubiquinol-8 and four-electron reduction of O2 to water. The one-electron reduced semiquinone forms transiently during the reaction, and the enzyme has been demonstrated to stabilize the semiquinone. The semiquinone is also formed in the D75E mutant, where the mutation has little influence on the catalytic activity, and in the D75H mutant, which is virtually inactive. In this work, wild-type cytochrome bo3 as well as the D75E and D75H mutant proteins were prepared with ubiquinone-8 13C-labeled selectively at the methyl and two methoxy groups. This was accomplished by expressing the proteins in a methionine auxotroph in the presence of l-methionine with the side chain methyl group 13C-labeled. The 13C-labeled quinone isolated from cytochrome bo3 was also used for the generation of model anion radicals in alcohol. Two-dimensional pulsed EPR and ENDOR were used for the study of the 13C methyl and methoxy hyperfine couplings in the semiquinone generated in the three proteins indicated above and in the model system. The data were used to characterize the transferred unpaired spin densities on the methyl and methoxy substituents and the conformations of the methoxy groups. In the wild type and D75E mutant, the constraints on the configurations of the methoxy side chains are similar, but the D75H mutant appears to have altered methoxy configurations, which could be related to the perturbed electron distribution in the semiquinone and the loss of enzymatic activity.

scholarly journals Substrate Specificity and Signal Transduction Pathways in the Glucose-Specific Enzyme II (EIIGlc) Component of the Escherichia coli Phosphotransferase System

2000 ◽  
Vol 182 (16) ◽  
pp. 4437-4442 ◽  
Author(s):  
Lucinda Notley-McRobb ◽  
Thomas Ferenci
Keyword(s):  
Escherichia Coli ◽  
Signal Transduction ◽  
Substrate Specificity ◽  
Dependent Manner ◽  
Wild Type ◽  
Specific Enzyme ◽  
Phosphotransferase System ◽  
Transmembrane Segments ◽  
In The Wild ◽  
Second Category

ABSTRACT Escherichia coli adapted to glucose-limited chemostats contained mutations in ptsG resulting in V12G, V12F, and G13C substitutions in glucose-specific enzyme II (EIIGlc) and resulting in increased transport of glucose and methyl-α-glucoside. The mutations also resulted in faster growth on mannose and glucosamine in a PtsG-dependent manner. By use of enhanced growth on glucosamine for selection, four further sites were identified where substitutions caused broadened substrate specificity (G176D, A288V, G320S, and P384R). The altered amino acids include residues previously identified as changing the uptake of ribose, fructose, and mannitol. The mutations belonged to two classes. First, at two sites, changes affected transmembrane residues (A288V and G320S), probably altering sugar selectivity directly. More remarkably, the five other specificity mutations affected residues unlikely to be in transmembrane segments and were additionally associated with increasedptsG transcription in the absence of glucose. Increased expression of wild-type EIIGlc was not by itself sufficient for growth with other sugars. A model is proposed in which the protein conformation determining sugar accessibility is linked to transcriptional signal transduction in EIIGlc. The conformation of EIIGlc elicited by either glucose transport in the wild-type protein or permanently altered conformation in the second category of mutants results in altered signal transduction and interaction with a regulator, probably Mlc, controlling the transcription of pts genes.

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