scholarly journals Escherichia coli TehB RequiresS-Adenosylmethionine as a Cofactor To Mediate Tellurite Resistance

2000 ◽  
Vol 182 (22) ◽  
pp. 6509-6513 ◽  
Author(s):  
Mingfu Liu ◽  
Raymond J. Turner ◽  
Tara L. Winstone ◽  
Andrea Saetre ◽  
Melanie Dyllick-Brenzinger ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nucleic Acid ◽  
Conformational Changes ◽  
Hydrodynamic Radius ◽  
Multicopy Plasmid ◽  
Cytoplasmic Protein ◽  
Wild Type ◽  
Methyltransferase Activity ◽  
Tellurite Resistance ◽  
Aspartate Residue

ABSTRACT The Escherichia coli chromosomal determinant for tellurite resistance consists of two genes (tehA andtehB) which, when expressed on a multicopy plasmid, confer resistance to K2TeO3 at 128 μg/ml, compared to the MIC of 2 μg/ml for the wild type. TehB is a cytoplasmic protein which possesses three conserved motifs (I, II, and III) found in S-adenosyl-l-methionine (SAM)-dependent non-nucleic acid methyltransferases. Replacement of the conserved aspartate residue in motif I by asparagine or alanine, or of the conserved phenylalanine in motif II by tyrosine or alanine, decreased resistance to background levels. Our results are consistent with motifs I and II in TehB being involved in SAM binding. Additionally, conformational changes in TehB are observed upon binding of both tellurite and SAM. The hydrodynamic radius of TehB measured by dynamic light scattering showed a ∼20% decrease upon binding of both tellurite and SAM. These data suggest that TehB utilizes a methyltransferase activity in the detoxification of tellurite.

scholarly journals 16S rRNA Mutations That Confer Tetracycline Resistance in Helicobacter pylori Decrease Drug Binding in Escherichia coli Ribosomes

2005 ◽  
Vol 187 (11) ◽  
pp. 3708-3712 ◽  
Author(s):  
Lisa Nonaka ◽  
Sean R. Connell ◽  
Diane E. Taylor
Keyword(s):  
Escherichia Coli ◽  
Helicobacter Pylori ◽  
16S Rrna ◽  
Binding Site ◽  
Tetracycline Resistance ◽  
Drug Binding ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
E Coli ◽  
H Pylori

ABSTRACT Tetracycline resistance in clinical isolates of Helicobacter pylori has been associated with nucleotide substitutions at positions 965 to 967 in the 16S rRNA. We constructed mutants which had different sequences at 965 to 967 in the 16S rRNA gene present on a multicopy plasmid in Escherichia coli strain TA527, in which all seven rrn genes were deleted. The MICs for tetracycline of all mutants having single, double, or triple substitutions at the 965 to 967 region that were previously found in highly resistant H. pylori isolates were higher than that of the mutant exhibiting the wild-type sequence of tetracycline-susceptible H. pylori. The MIC of the mutant with the 965TTC967 triple substitution was 32 times higher than that of the E. coli mutant with the 965AGA967 substitution present in wild-type H. pylori. The ribosomes extracted from the tetracycline-resistant E. coli 965TTC967 variant bound less tetracycline than E. coli with the wild-type H. pylori sequence at this region. The concentration of tetracycline bound to the ribosome was 40% that of the wild type. The results of this study suggest that tetracycline binding to the primary binding site (Tet-1) of the ribosome at positions 965 to 967 is influenced by its sequence patterns, which form the primary binding site for tetracycline.

scholarly journals Mechanism of thermal denaturation of maltodextrin phosphorylase from Escherichia coli

2000 ◽  
Vol 346 (2) ◽  
pp. 255-263 ◽  
Author(s):  
Richard GRIEßLER ◽  
Sabato D'AURIA ◽  
Reinhard SCHINZEL ◽  
Fabio TANFANI ◽  
Bernd NIDETZKY
Keyword(s):  
Escherichia Coli ◽  
Secondary Structure ◽  
Conformational Changes ◽  
Active Site ◽  
Thermal Denaturation ◽  
Hydrophobic Interactions ◽  
Wild Type ◽  
Reversible Inactivation ◽  
The Stability ◽  
Maltodextrin Phosphorylase

Maltodextrin phosphorylase from Escherichia coli (MalP) is a dimeric protein in which each ≈ 90-kDa subunit contains active-site pyridoxal 5ʹ-phosphate. To unravel factors contributing to the stability of MalP, thermal denaturations of wild-type MalP and a thermostable active-site mutant (Asn-133 → Ala) were compared by monitoring enzyme activity, cofactor dissociation, secondary structure content and aggregation. Small structural transitions of MalP are shown by Fourier-transform infrared spectroscopy to take place at ≈ 45 °C. They are manifested by slight increases in unordered structure and 1H/2H exchange, and reflect reversible inactivation of MalP. Aggregation of the MalP dimer is triggered by these conformational changes and starts at ≈ 45 °C without prior release into solution of pyridoxal 5ʹ-phosphate. It is driven by electrostatic rather than hydrophobic interactions between MalP dimers, and leads to irreversible inactivation of the enzyme. Aggregation is inhibited efficiently and specifically by oxyanions such as phosphate, and AMP which therefore, stabilize MalP against the irreversible denaturation step at 45 °C. Melting of the secondary structure in soluble and aggregated MalP takes place at much higher temperatures of approx. 58 and 67 °C, respectively. Replacement of Asn-133 by Ala does not change the mechanism of thermal denaturation, but leads to a shift of the entire pathway to a ≈ 15 °C higher value on the temperature scale. Apart from greater stability, the Asn-133 → Ala mutant shows a 2-fold smaller turnover number and a 4.6-fold smaller energy of activation than wild-type MalP, probably indicating that the site-specific replacement of Asn-133 brings about a greater rigidity of the active-site environment of the enzyme. A structure-based model is proposed which explains the stabilizing interaction between MalP and oxyanions, or AMP.

scholarly journals Mutations in Residues Involved in Zinc Binding in the Catalytic Site of Escherichia coli Threonyl-tRNA Synthetase Confer a Dominant Lethal Phenotype

2007 ◽  
Vol 189 (19) ◽  
pp. 6839-6848 ◽  
Author(s):  
Joël Caillet ◽  
Monique Graffe ◽  
Flore Eyermann ◽  
Pascale Romby ◽  
Mathias Springer
Keyword(s):  
Escherichia Coli ◽  
Trna Synthetase ◽  
Dominant Negative ◽  
Zinc Binding ◽  
Regulatory Function ◽  
Dominant Negative Effect ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
Zinc Binding Site ◽  
Negative Effect

ABSTRACT Escherichia coli threonyl-tRNA synthetase is a homodimeric protein that acts as both an enzyme and a regulator of gene expression: the protein aminoacylates tRNAThr isoacceptors and binds to its own mRNA, inhibiting its translation. The enzyme contains a zinc atom in its active site, which is essential for the recognition of threonine. Mutations in any of the three amino acids forming the zinc-binding site inactivate the enzyme and have a dominant negative effect on growth if the corresponding genes are placed on a multicopy plasmid. We show here that this particular property is not due to the formation of inactive heterodimers, the titration of tRNAThr by an inactive enzyme, or its misaminoacylation but is, rather, due to the regulatory function of threonyl-tRNA synthetase. Overproduction of the inactive enzyme represses the expression of the wild-type chromosomal copy of the gene to an extent incompatible with bacterial growth.

mraW, an essential gene at the dcw cluster of Escherichia coli codes for a cytoplasmic protein with methyltransferase activity

Biochimie ◽  
1999 ◽  
Vol 81 (8-9) ◽  
pp. 879-888 ◽  
Author(s):  
Maite Carrión ◽  
Manuel J. Gómez ◽  
Rafael Merchante-Schubert ◽  
Silvina Dongarrá ◽  
Juan A. Ayala
Keyword(s):  
Escherichia Coli ◽  
Essential Gene ◽  
Cytoplasmic Protein ◽  
Methyltransferase Activity

scholarly journals Construction and Initial Characterization of Escherichia coli Strains with Few or No Intact Chromosomal rRNA Operons

1999 ◽  
Vol 181 (12) ◽  
pp. 3803-3809 ◽  
Author(s):  
Tsuneaki Asai ◽  
Ciarán Condon ◽  
Justina Voulgaris ◽  
Dmitry Zaporojets ◽  
Binghua Shen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Rrna Operon ◽  
Rrna Genes ◽  
23S Rrna ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
Protein Ratio ◽  
23S Rrna Genes

ABSTRACT The Escherichia coli genome carries seven rRNA (rrn) operons, each containing three rRNA genes. The presence of multiple operons has been an obstacle to many studies of rRNA because the effect of mutations in one operon is diluted by the six remaining wild-type copies. To create a tool useful for manipulating rRNA, we sequentially inactivated from one to all seven of these operons with deletions spanning the 16S and 23S rRNA genes. In the final strain, carrying no intact rRNA operon on the chromosome, rRNA molecules were expressed from a multicopy plasmid containing a single rRNA operon (prrn). Characterization of these rrndeletion strains revealed that deletion of two operons was required to observe a reduction in the growth rate and rRNA/protein ratio. When the number of deletions was extended from three to six, the decrease in the growth rate was slightly more than the decrease in the rRNA/protein ratio, suggesting that ribosome efficiency was reduced. This reduction was most pronounced in the Δ7 prrn strain, in which the growth rate, unlike the rRNA/protein ratio, was not completely restored to wild-type levels by a cloned rRNA operon. The decreases in growth rate and rRNA/protein ratio were surprisingly moderate in the rrndeletion strains; the presence of even a single operon on the chromosome was able to produce as much as 56% of wild-type levels of rRNA. We discuss possible applications of these strains in rRNA studies.

scholarly journals Function of the ςE Regulon in Dead-Cell Lysis in Stationary-Phase Escherichia coli

2000 ◽  
Vol 182 (18) ◽  
pp. 5231-5237 ◽  
Author(s):  
Takeshi Nitta ◽  
Hiroshi Nagamitsu ◽  
Masayuki Murata ◽  
Hanae Izu ◽  
Mamoru Yamada
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Cell Lysis ◽  
Dead Cell ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
E Coli ◽  
Intracellular Proteins ◽  
In The Wild ◽  
Cloned Gene

ABSTRACT Elevation of active ςE levels in Escherichia coli by either repressing the expression of rseAencoding an anti-ςE factor or cloning rpoE in a multicopy plasmid, led to a large decrease in the number of dead cells and the accumulation of cellular proteins in the medium in the stationary phase. The numbers of CFU, however, were nearly the same as those of the wild type or cells devoid of the cloned gene. In the wild-type cells, rpoE expression was increased in the stationary phase and a low-level release of intracellular proteins was observed. These results suggest that dead cell lysis in stationary-phase E. coli occurs in a ςE-dependent fashion. We propose there is a novel physiological function of the ςE regulon that may guarantee cell survival in prolonged stationary phase by providing nutrients from dead cells for the next generation.

scholarly journals Glucose-6-phosphate dehydrogenase and ferredoxin-NADP(H) reductase contribute to damage repair during the soxRS response of Escherichia coli

Microbiology ◽  
2006 ◽  
Vol 152 (4) ◽  
pp. 1119-1128 ◽  
Author(s):  
Mariana Giró ◽  
Néstor Carrillo ◽  
Adriana R. Krapp
Keyword(s):  
Escherichia Coli ◽  
Survival Rates ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
Iron Sulfur Clusters ◽  
Iron Sulfur ◽  
Oxidative Challenge ◽  
Glucose 6 Phosphate Dehydrogenase

The NADP(H)-dependent enzymes glucose-6-phosphate dehydrogenase (G6PDH) and ferredoxin(flavodoxin)-NADP(H) reductase (FPR), encoded by the zwf and fpr genes, respectively, are committed members of the soxRS regulatory system involved in superoxide resistance in Escherichia coli. Exposure of E. coli cells to the superoxide propagator methyl viologen (MV) led to rapid accumulation of G6PDH, while FPR was induced after a lag period of several minutes. Bacteria expressing G6PDH from a multicopy plasmid accumulated higher NADPH levels and displayed a protracted soxRS response, whereas FPR build-up had the opposite effects. Inactivation of either of the two genes resulted in enhanced sensitivity to MV killing, while further increases in the cellular content of FPR led to higher survival rates under oxidative conditions. In contrast, G6PDH accumulation over wild-type levels of expression failed to increase MV tolerance. G6PDH and FPR could act concertedly to deliver reducing equivalents from carbohydrates, via NADP+, to the FPR acceptors ferredoxin and/or flavodoxin. To evaluate whether this electron-transport system could mediate reductive repair reactions, the pathway was reconstituted in vitro from purified components; the reconstituted system was found to be functional in reactivation of oxidatively damaged iron–sulfur clusters of hydro-lyases such as aconitase and 6-phosphogluconate dehydratase. Recovery of these activities after oxidative challenge was faster and more extensive in transformed bacteria overexpressing FPR than in wild-type cells, indicating that the reductase could sustain hydro-lyase repair in vivo. However, FPR-deficient mutants were still able to fix iron–sulfur clusters at significant rates, suggesting that back-up routes for ferredoxin and/or flavodoxin reduction might be called into action to rescue inactivated enzymes when FPR is absent.

scholarly journals Deletion and Substitution Analysis of the Escherichia coli TonB Q160 Region

2007 ◽  
Vol 189 (13) ◽  
pp. 4662-4670 ◽  
Author(s):  
Hema Vakharia-Rao ◽  
Kyle A. Kastead ◽  
Marina I. Savenkova ◽  
Charles M. Bulathsinghala ◽  
Kathleen Postle
Keyword(s):  
Escherichia Coli ◽  
Conformational Changes ◽  
Vitamin B ◽  
Initial Contact ◽  
Wild Type ◽  
Carboxy Terminus ◽  
Mutant Proteins ◽  
Nested Deletions ◽  
Energy Dependent ◽  
Arginyl Residues

ABSTRACT The active transport of iron siderophores and vitamin B12 across the outer membrane (OM) of Escherichia coli requires OM transporters and the potential energy of the cytoplasmic membrane (CM) proton gradient and CM proteins TonB, ExbB, and ExbD. A region at the amino terminus of the transporter, called the TonB box, directly interacts with TonB Q160 region residues. R158 and R166 in the TonB Q160 region were proposed to play important roles in cocrystal structures of the TonB carboxy terminus with OM transporters BtuB and FhuA. In contrast to predictions based on the crystal structures, none of the single, double, or triple alanyl substitutions at arginyl residues significantly decreased TonB activity. Even the quadruple R154A R158A R166A R171A mutant TonB still retained 30% of wild-type activity. Up to five residues centered on TonB Q160 could be deleted without inactivating TonB or preventing its association with the OM. TonB mutant proteins with nested deletions of 7, 9, or 11 residues centered on TonB Q160 were inactive and appeared never to have associated with the OM. Because the 7-residue-deletion mutant protein (TonBΔ7, lacking residues S157 to Y163) could still form disulfide-linked dimers when combined with W213C or F202C in the TonB carboxy terminus, the TonBΔ7 deletion did not prevent necessary energy-dependent conformational changes that occur in the CM. Thus, it appeared that initial contact with the OM is made through TonB residues S157 to Y163. It is hypothesized that the TonB Q160 region may be part of a large disordered region required to span the periplasm and contact an OM transporter.

scholarly journals SoxRS-Regulated Expression and Genetic Analysis of the yggX Gene of Escherichia coli

2003 ◽  
Vol 185 (22) ◽  
pp. 6624-6632 ◽  
Author(s):  
Pablo J. Pomposiello ◽  
Anastasia Koutsolioutsou ◽  
Daniel Carrasco ◽  
Bruce Demple
Keyword(s):  
Oxidative Stress ◽  
Escherichia Coli ◽  
Oxidative Damage ◽  
Type Strain ◽  
Wild Type Strain ◽  
Ectopic Expression ◽  
Deletion Strain ◽  
Northern Analysis ◽  
Multicopy Plasmid ◽  
Wild Type

ABSTRACT Genomic studies with bacteria have identified redox-responsive genes without known roles in counteracting oxidative damage. Previous transcriptional profiling showed that expression of one such gene, yggX, was activated by superoxide stress in Escherichia coli. Here we show that this activation could be mimicked by artificial expression of the regulatory protein SoxS. Northern analysis confirmed the transcriptional activation of yggX by oxidative stress or SoxS expression but not in response to the related MarA or Rob proteins. Northern analysis showed that mltC, which codes for a peptidoglycan hydrolase and is positioned immediately downstream of yggX, was also regulated by oxidative stress or ectopic expression of SoxS. Purified SoxS protein bound to the predicted yggX promoter region, between positions 223 and 163 upstream from the yggX translational start site. Within this region, a 20-bp sequence was found to be necessary for oxidative stress-mediated activation of yggX transcription. A yggX deletion strain was hypersensitive to the redox-cycling agent paraquat, and a plasmid expressing YggX complemented the sensitivity of the deletion strain. Under exposure to paraquat, the yggX deletion strain showed a deficiency in aconitase activity compared to the isogenic wild-type strain, while expression of YggX from a multicopy plasmid increased the aconitase levels above those of the wild-type strain. These results demonstrate the direct regulation of the yggX gene by the redox-sensing SoxRS system and provide further evidence for the involvement of yggX in protection of iron-sulfur proteins against oxidative damage.

scholarly journals NEGATIVE DOMINANT MUTATIONS OF THE uidR GENE IN ESCHERICHIA COLI: GENETIC PROOF FOR A COOPERATIVE REGULATION OF uidA EXPRESSION

Genetics ◽  
1986 ◽  
Vol 112 (2) ◽  
pp. 173-182
Author(s):  
Carlos Blanco ◽  
Paul Ritzenthaler ◽  
Mireille Mata-Gilsinger
Keyword(s):  
Escherichia Coli ◽  
Gene Dosage ◽  
Regulatory Gene ◽  
Active Form ◽  
Point Mutations ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
Cooperative Process ◽  
Mutant Phenotypes ◽  
K 12

ABSTRACT The uidA gene is the first gene involved in the hexuronide-hexuronate pathway in Escherichia coli K-12 and is under the dual control of the uidR and uxuR encoded repressors. Point mutations affecting the uidR regulatory gene were sought to investigate the regulation of uidA. When the uidR mutant allele was on a multicopy plasmid and the wild-type allele was on the chromosome, some of the mutant phenotypes were dominant to the wild-type phenotype, indicating that the active form of the UidR repressor is multimeric. We have demonstrated that expression of the mutant phenotype is dependent on gene dosage. The dominance of the uidR allele was also sensitive to the presence of the wild-type uxuR allele in the cell. This behavior probably results from UidR-UxuR repressor interactions. A mechanism is proposed: we suggest that the UidR and UxuR repressors interact after their binding to the operator site of uidA; the binding of one regulatory molecule may facilitate the binding of the other one in a cooperative process.

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