scholarly journals MazG, a Nucleoside Triphosphate Pyrophosphohydrolase, Interacts with Era, an Essential GTPase in Escherichia coli

2002 ◽  
Vol 184 (19) ◽  
pp. 5323-5329 ◽  
Author(s):  
Junjie Zhang ◽  
Masayori Inouye
Keyword(s):  
Escherichia Coli ◽  
Direct Interaction ◽  
Kanamycin Resistance ◽  
Nucleoside Triphosphate ◽  
Cellular Functions ◽  
Genomic Libraries ◽  
E Coli ◽  
Yeast Two Hybrid System ◽  
Nucleoside Triphosphate Pyrophosphohydrolase

ABSTRACT Era is an essential GTPase in Escherichia coli, and Era has been implicated in a number of cellular functions. Homologues of Era have been identified in various bacteria and some eukaryotes. Using the era gene as bait in the yeast two-hybrid system to screen E. coli genomic libraries, we discovered that Era interacts with MazG, a protein of unknown function which is highly conserved among bacteria. The direct interaction between Era and MazG was also confirmed in vitro, being stronger in the presence of GDP than in the presence of GTPγS. MazG was characterized as a nucleoside triphosphate pyrophosphohydrolase which can hydrolyze all eight of the canonical ribo- and deoxynucleoside triphosphates to their respective monophosphates and PPi, with a preference for deoxynucleotides. A mazG deletion strain of E. coli was constructed by replacing the mazG gene with a kanamycin resistance gene. Unlike mutT, a gene for another conserved nucleotide triphosphate pyrophosphohydrolase that functions as a mutator gene, the mazG deletion did not result in a mutator phenotype in E. coli.

scholarly journals 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

2008 ◽  
Vol 190 (10) ◽  
pp. 3434-3443 ◽  
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.

scholarly journals Excision of the doubly methylated base N 4 ,5-dimethylcytosine from DNA by Escherichia coli Nei and Fpg proteins

2018 ◽  
Vol 373 (1748) ◽  
pp. 20170337 ◽  
Author(s):  
Marina Alexeeva ◽  
Prashanna Guragain ◽  
Almaz N. Tesfahun ◽  
Miglė Tomkuvienė ◽  
Aysha Arshad ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Alkylating Agents ◽  
High Sensitivity ◽  
Cellular Functions ◽  
Repair Enzyme ◽  
E Coli ◽  
Chemical Evidence ◽  
Adenine Thymine

Cytosine (C) in DNA is often modified to 5-methylcytosine (m 5 C) to execute important cellular functions. Despite the significance of m 5 C for epigenetic regulation in mammals, damage to m 5 C has received little attention. For instance, almost no studies exist on erroneous methylation of m 5 C by alkylating agents to doubly or triply methylated bases. Owing to chemical evidence, and because many prokaryotes express methyltransferases able to convert m 5 C into N 4 ,5-dimethylcytosine (m N 4,5 C) in DNA, m N 4,5 C is probably present in vivo . We screened a series of glycosylases from prokaryotic to human and found significant DNA incision activity of the Escherichia coli Nei and Fpg proteins at m N 4,5 C residues in vitro . The activity of Nei was highest opposite cognate guanine followed by adenine, thymine (T) and C. Fpg-complemented Nei by exhibiting the highest activity opposite C followed by lower activity opposite T. To our knowledge, this is the first description of a repair enzyme activity at a further methylated m 5 C in DNA, as well as the first alkylated base allocated as a Nei or Fpg substrate. Based on our observed high sensitivity to nuclease S1 digestion, we suggest that m N 4,5 C occurs as a disturbing lesion in DNA and that Nei may serve as a major DNA glycosylase in E. coli to initiate its repair. This article is part of a discussion meeting issue ‘Frontiers in epigenetic chemical biology’.

scholarly journals Regulated Expression of the Escherichia coli lepB Gene as a Tool for Cellular Testing of Antimicrobial Compounds That Inhibit Signal Peptidase I In Vitro

2002 ◽  
Vol 46 (11) ◽  
pp. 3549-3554 ◽  
Author(s):  
Maria D. F. S. Barbosa ◽  
Siqi Lin ◽  
Jay A. Markwalder ◽  
Jonathan A. Mills ◽  
Joseph A. DeVito ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Activity ◽  
Polymyxin B ◽  
Kanamycin Resistance ◽  
Signal Peptidase ◽  
Prolonged Incubation ◽  
E Coli ◽  
Regulated Expression ◽  
Signal Peptidase I

ABSTRACT Escherichia coli under-expressing lepB was utilized to test cellular inhibition of signal peptidase I (SPase). For the construction of a lepB regulatable strain, the E. coli lepB gene was cloned into pBAD, with expression dependent on l-arabinose. The chromosomal copy of lepB was replaced with a kanamycin resistance gene, which was subsequently removed. SPase production by the lepB regulatable strain in the presence of various concentrations of l-arabinose was monitored by Western blot analysis. At lower arabinose concentrations growth proceeded more slowly, possibly due to a decrease of SPase levels in the cells. A penem SPase inhibitor with little antimicrobial activity against E. coli when tested at 100 μM was utilized to validate the cell-based system. Under-expression of lepB sensitized the cells to penem, with complete growth inhibition observed at 10 to 30 μM. Growth was rescued by increasing the SPase levels. The cell-based assay was used to test cellular inhibition of SPase by compounds that inhibit the enzyme in vitro. MD1, MD2, and MD3 are SPase inhibitors with antimicrobial activity against Staphylococcus aureus, although they do not inhibit growth of E. coli. MD1 presented the best spectrum of antimicrobial activity. Both MD1 and MD2 prevented growth of E. coli under-expressing lepB in the presence of polymyxin B nonapeptide, with growth rescue observed when wild-type levels of SPase were produced. MD3 and MD4, a reactive analog of MD3, inhibited growth of E. coli under-expressing lepB. However, growth rescue in the presence of these compounds following increased lepB expression was observed only after prolonged incubation.

Substituted 4-Formyl-2H-chromen-2-ones: Their Reaction with N-(2,3,4,6-Tetra-Oacetyl- β-D-galactopyranosyl)thiosemicarbazide, Antibacterial and Antifungal Activity of Their Thiosemicarbazone Products

2020 ◽  
Vol 24 (19) ◽  
pp. 2272-2282
Author(s):  
Vu Ngoc Toan ◽  
Nguyen Minh Tri ◽  
Nguyen Dinh Thanh
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Candida Albicans ◽  
Selenium Dioxide ◽  
Antibacterial And Antifungal Activity ◽  
E Coli ◽  
Antibacterial And Antifungal Activities ◽  
Alkyl Ethers ◽  
Antibacterial And Antifungal

Several 6- and 7-alkoxy-2-oxo-2H-chromene-4-carbaldehydes were prepared from corresponding alkyl ethers of 6- and 7-hydroxy-4-methyl-2-oxo-2H-chromen-2-ones by oxidation using selenium dioxide. 6- and 7-Alkoxy-4-methyl-2H-chromenes were obtained with yields of 57-85%. Corresponding 4-carbaldehyde derivatives were prepared with yields of 41-67%. Thiosemicarbazones of these aldehydes with D-galactose moiety were synthesized by reaction of these aldehydes with N-(2,3,4,6-tetra-O-acetyl-β-Dgalactopyranosyl) thiosemicarbazide with yields of 62-74%. These thiosemicarbazones were screened for their antibacterial and antifungal activities in vitro against bacteria, such as Staphylococcus aureus, Escherichia coli, and fungi, such as Aspergillus niger, Candida albicans. Several compounds exhibited strong inhibitory activity with MIC values of 0.78- 1.56 μM, including 8a (against S. aureus, E. coli, and C. albicans), 8d (against E. coli and A. niger), 9a (against S. aureus), and 9c (against S. aureus and C. albicans).

scholarly journals In vitro activity of antimicrobial peptide CDP-B11 alone and in combination with colistin against colistin-resistant and multidrug-resistant Escherichia coli

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kaitlin S. Witherell ◽  
Jason Price ◽  
Ashok D. Bandaranayake ◽  
James Olson ◽  
Douglas R. Call
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Peptide ◽  
Membrane Integrity ◽  
Antibiotic Resistance Genes ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Multidrug Resistant Bacteria ◽  
E Coli ◽  
Minimal Media

AbstractMultidrug-resistant bacteria are a growing global concern, and with increasingly prevalent resistance to last line antibiotics such as colistin, it is imperative that alternative treatment options are identified. Herein we investigated the mechanism of action of a novel antimicrobial peptide (CDP-B11) and its effectiveness against multidrug-resistant bacteria including Escherichia coli #0346, which harbors multiple antibiotic-resistance genes, including mobilized colistin resistance gene (mcr-1). Bacterial membrane potential and membrane integrity assays, measured by flow cytometry, were used to test membrane disruption. Bacterial growth inhibition assays and time to kill assays measured the effectiveness of CDP-B11 alone and in combination with colistin against E. coli #0346 and other bacteria. Hemolysis assays were used to quantify the hemolytic effects of CDP-B11 alone and in combination with colistin. Findings show CDP-B11 disrupts the outer membrane of E. coli #0346. CDP-B11 with colistin inhibits the growth of E. coli #0346 at ≥ 10× lower colistin concentrations compared to colistin alone in Mueller–Hinton media and M9 media. Growth is significantly inhibited in other clinically relevant strains, such as Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. In rich media and minimal media, the drug combination kills bacteria at a lower colistin concentration (1.25 μg/mL) compared to colistin alone (2.5 μg/mL). In minimal media, the combination is bactericidal with killing accelerated by up to 2 h compared to colistin alone. Importantly, no significant red blood hemolysis is evident for CDP-B11 alone or in combination with colistin. The characteristics of CDP-B11 presented here indicate that it can be used as a potential monotherapy or as combination therapy with colistin for the treatment of multidrug-resistant infections, including colistin-resistant infections.

scholarly journals Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product

1987 ◽  
Vol 248 (1) ◽  
pp. 43-51 ◽  
Author(s):  
J Charlier ◽  
R Sanchez
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Activity Ratio ◽  
Deae Cellulose ◽  
Trna Synthetase ◽  
Trna Synthetases ◽  
E Coli ◽  
Aminoacyl Trna Synthetases

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5′)tetraphospho(5′)adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

scholarly journals A Rhodobacter sphaeroides Protein Mechanistically Similar to Escherichia coli DksA Regulates Photosynthetic Growth

mBio ◽  
2014 ◽  
Vol 5 (3) ◽  
Author(s):  
Christopher W. Lennon ◽  
Kimberly C. Lemmer ◽  
Jessica L. Irons ◽  
Max I. Sellman ◽  
Timothy J. Donohue ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Structure Function ◽  
Rhodobacter Sphaeroides ◽  
Fatty Acid Content ◽  
Regulatory Protein ◽  
Growth Conditions ◽  
Globular Domain ◽  
Content Type ◽  
E Coli

ABSTRACTDksA is a global regulatory protein that, together with the alarmone ppGpp, is required for the “stringent response” to nutrient starvation in the gammaproteobacteriumEscherichia coliand for more moderate shifts between growth conditions. DksA modulates the expression of hundreds of genes, directly or indirectly. Mutants lacking a DksA homolog exhibit pleiotropic phenotypes in other gammaproteobacteria as well. Here we analyzed the DksA homolog RSP2654 in the more distantly relatedRhodobacter sphaeroides, an alphaproteobacterium. RSP2654 is 42% identical and similar in length toE. coliDksA but lacks the Zn finger motif of theE. coliDksA globular domain. Deletion of the RSP2654 gene results in defects in photosynthetic growth, impaired utilization of amino acids, and an increase in fatty acid content. RSP2654 complements the growth and regulatory defects of anE. colistrain lacking thedksAgene and modulates transcriptionin vitrowithE. coliRNA polymerase (RNAP) similarly toE. coliDksA. RSP2654 reduces RNAP-promoter complex stabilityin vitrowith RNAPs fromE. coliorR. sphaeroides, alone and synergistically with ppGpp, suggesting that even though it has limited sequence identity toE. coliDksA (DksAEc), it functions in a mechanistically similar manner. We therefore designate the RSP2654 protein DksARsp. Our work suggests that DksARsphas distinct and important physiological roles in alphaproteobacteria and will be useful for understanding structure-function relationships in DksA and the mechanism of synergy between DksA and ppGpp.IMPORTANCEThe role of DksA has been analyzed primarily in the gammaproteobacteria, in which it is best understood for its role in control of the synthesis of the translation apparatus and amino acid biosynthesis. Our work suggests that DksA plays distinct and important physiological roles in alphaproteobacteria, including the control of photosynthesis inRhodobacter sphaeroides. The study of DksARsp, should be useful for understanding structure-function relationships in the protein, including those that play a role in the little-understood synergy between DksA and ppGpp.

scholarly journals Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

2005 ◽  
Vol 71 (7) ◽  
pp. 3468-3474 ◽  
Author(s):  
Gyeong Tae Eom ◽  
Jae Kwang Song ◽  
Jung Hoon Ahn ◽  
Yeon Soo Seo ◽  
Joon Shick Rhee
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Abc Transporter ◽  
Microtiter Plate ◽  
Single Amino Acid ◽  
Wild Type ◽  
E Coli ◽  
Single Amino Acid Change ◽  
Secretion Efficiency

ABSTRACT The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.

scholarly journals SpoVID Guides SafA to the Spore Coat inBacillus subtilis

2001 ◽  
Vol 183 (10) ◽  
pp. 3041-3049 ◽  
Author(s):  
Amanda J. Ozin ◽  
Craig S. Samford ◽  
Adriano O. Henriques ◽  
Charles P. Moran
Keyword(s):  
Direct Interaction ◽  
Spore Coat ◽  
Coat Proteins ◽  
Yeast Two Hybrid System ◽  
Spore Coat Proteins ◽  
Basement Layer ◽  
Two Hybrid ◽  
Spore Coat Protein

ABSTRACT Bacteria assemble complex structures by targeting proteins to specific subcellular locations. The protein coat that encasesBacillus subtilis spores is an example of a structure that requires coordinated targeting and assembly of more than 24 polypeptides. The earliest stages of coat assembly require the action of three morphogenetic proteins: SpoIVA, CotE, and SpoVID. In the first steps, a basement layer of SpoIVA forms around the surface of the forespore, guiding the subsequent positioning of a ring of CotE protein about 75 nm from the forespore surface. SpoVID localizes near the forespore membrane where it functions to maintain the integrity of the CotE ring and to anchor the nascent coat to the underlying spore structures. However, it is not known which spore coat proteins interact directly with SpoVID. In this study we examined the interaction between SpoVID and another spore coat protein, SafA, in vivo using the yeast two-hybrid system and in vitro. We found evidence that SpoVID and SafA directly interact and that SafA interacts with itself. Immunofluorescence microscopy showed that SafA localized around the forespore early during coat assembly and that this localization of SafA was dependent on SpoVID. Moreover, targeting of SafA to the forespore was also dependent on SpoIVA, as was targeting of SpoVID to the forespore. We suggest that the localization of SafA to the spore coat requires direct interaction with SpoVID.

scholarly journals σE Regulates and Is Regulated by a Small RNA in Escherichia coli

2007 ◽  
Vol 189 (11) ◽  
pp. 4243-4256 ◽  
Author(s):  
Karl M. Thompson ◽  
Virgil A. Rhodius ◽  
Susan Gottesman
Keyword(s):  
Escherichia Coli ◽  
Small Rnas ◽  
Noncoding Rna ◽  
Response Regulator ◽  
Heterologous Promoter ◽  
Genomic Libraries ◽  
Multicopy Suppressors ◽  
Intergenic Regions ◽  
Transposon Mutants

ABSTRACT RybB is a small, Hfq-binding noncoding RNA originally identified in a screen of conserved intergenic regions in Escherichia coli. Fusions of the rybB promoter to lacZ were used to screen plasmid genomic libraries and genomic transposon mutants for regulators of rybB expression. A number of plasmids, including some carrying rybB, negatively regulated the fusion. An insertion in the rep helicase and one upstream of dnaK decreased expression of the fusion. Multicopy suppressors of these insertions led to identification of two plasmids that stimulated the fusion. One contained the gene for the response regulator OmpR; the second contained mipA, encoding a murein hydrolase. The involvement of MipA and OmpR in cell surface synthesis suggested that the rybB promoter might be dependent on σE. The sequence upstream of the +1 of rybB contains a consensus σE promoter. The activity of rybB-lacZ was increased in cells lacking the RseA anti-sigma factor and when σE was overproduced from a heterologous promoter. The activity of rybB-lacZ and the detection of RybB were totally abolished in an rpoE-null strain. In vitro, σE efficiently transcribes from this promoter. Both a rybB mutation and an hfq mutation significantly increased expression of both rybB-lacZ and rpoE-lacZ fusions, consistent with negative regulation of the σE response by RybB and other small RNAs. Based on the plasmid screens, NsrR, a repressor sensitive to nitric oxide, was also found to negatively regulate σE-dependent promoters in an RseA-independent fashion.

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