scholarly journals Characterization of Streptococcus pneumoniae TrmD, a tRNA Methyltransferase Essential for Growth

2004 ◽  
Vol 186 (8) ◽  
pp. 2346-2354 ◽  
Author(s):  
Karen O'Dwyer ◽  
Joseph M. Watts ◽  
Sanjoy Biswas ◽  
Jennifer Ambrad ◽  
Michael Barber ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Kinetic Studies ◽  
Mobility Shift ◽  
Regulation Of Expression ◽  
E Coli ◽  
Trna Species ◽  
Sequential Mechanism ◽  
Trna Methyltransferase ◽  
Gel Mobility Shift

ABSTRACT Down-regulation of expression of trmD, encoding the enzyme tRNA (guanosine-1)-methyltransferase, has shown that this gene is essential for growth of Streptococcus pneumoniae. The S. pneumoniae trmD gene has been isolated and expressed in Escherichia coli by using a His-tagged T7 expression vector. Recombinant protein has been purified, and its catalytic and physical properties have been characterized. The native enzyme displays a molecular mass of approximately 65,000 Da, suggesting that streptococcal TrmD is a dimer of two identical subunits. In fact, this characteristic can be extended to several other TrmD orthologs, including E. coli TrmD. Kinetic studies show that the streptococcal enzyme utilizes a sequential mechanism. Binding of tRNA by gel mobility shift assays gives a dissociation constant of 22 nM for one of its substrates, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathit{tRNA}_{\mathit{CAG}}^{\mathit{Leu}}\) \end{document} . Other heterologous nonsubstrate tRNA species, like \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathit{tRNA}_{\mathit{GGT}}^{\mathit{Thr}}\) \end{document} , tRNAPhe, and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathit{tRNA}_{\mathit{TGC}}^{\mathit{Ala}}\) \end{document} , bind the enzyme with similar affinities, suggesting that tRNA specificity is achieved via a postbinding event(s).

scholarly journals Regulation of Expression of theyiaKLMNOPQRS Operon for Carbohydrate Utilization inEscherichia coli: Involvement of the Main Transcriptional Factors

2000 ◽  
Vol 182 (16) ◽  
pp. 4617-4624 ◽  
Author(s):  
Ester Ibañez ◽  
Evangelina Campos ◽  
Laura Baldoma ◽  
Juan Aguilar ◽  
Josefa Badia
Keyword(s):  
Mutant Strain ◽  
Integration Host Factor ◽  
Anaerobic Growth ◽  
Receptor Protein ◽  
Mobility Shift ◽  
Regulation Of Expression ◽  
Carbohydrate Utilization ◽  
In The Wild ◽  
Gel Mobility Shift ◽  
Transcriptional Fusions

ABSTRACT The yiaKLMNOPQRS (yiaK-S) gene cluster ofEscherichia coli is believed to be involved in the utilization of a hitherto unknown carbohydrate which generates the intermediate l-xylulose. Transcription ofyiaK-S as a single message from the unique promoter found upstream of yiaK is proven in this study. The 5′ end has been located at 60 bp upstream from the ATG. Expression of theyiaK-S operon is controlled in the wild-type strain by a repressor encoded by yiaJ. No inducer molecule of theyiaK-S operon has been identified among over 80 carbohydrate or derivative compounds tested, the system being expressed only in a mutant strain lacking the YiaJ repressor. ThelacZ transcriptional fusions in the genetic background of the mutant strain revealed that yiaK-S is modulated by the integration host factor and by the cyclic AMP (cAMP)-cAMP receptor protein (Crp) activator complex. A twofold increase in the induction was observed during anaerobic growth, which was independent of ArcA or Fnr. Gel mobility shift assays showed that the YiaJ repressor binds to a promoter fragment extending from −50 to +121. These studies also showed that the cAMP-Crp complex can bind to two different sites. ThelacZ transcriptional fusions of different fragments of the promoter demonstrated that binding of cAMP-Crp to the Crp site 1, centered at −106, is essential for yiaK-S expression. The 5′ end of the yiaJ gene was determined, and its promoter region was found to overlap with the divergent yiaK-Spromoter. Expression of yiaJ is autogenously regulated and reduced by the binding of Crp-cAMP to the Crp site 1 of theyiaK-S promoter.

scholarly journals Characterization of a telomere-binding protein from Physarum polycephalum.

1991 ◽  
Vol 11 (4) ◽  
pp. 2282-2290 ◽  
Author(s):  
J S Coren ◽  
E M Epstein ◽  
V M Vogt
Keyword(s):  
Physarum Polycephalum ◽  
Nuclear Protein ◽  
Binding Activity ◽  
Binding Assays ◽  
Mobility Shift ◽  
Heat Stable ◽  
Dnase I Footprinting ◽  
Telomere Binding Protein ◽  
Gel Mobility Shift

We have partially purified a nuclear protein (PPT) from Physarum polycephalum that binds to the extrachromosomal ribosomal DNA telomeres of this acellular slime mold. Binding is specific for the (T2AG3)n telomere repeats, as evidenced by nitrocellulose filter binding assays, by gel mobility shift assays with both DNA fragments and double-stranded oligonucleotides, and by DNase I footprinting. PPT is remarkably heat stable, showing undiminished binding activity after incubation at 90 degrees C. It sediments at 1.2S, corresponding to a molecular weight of about 10,000 (for a globular protein), and its binding activity is undiminished by incubation with RNase, suggesting that it is not a ribonucleoprotein. We hypothesize that PPT plays a structural role in telomeres, perhaps preventing nucleolytic degradation or promoting telomere extension by a telomere-specific terminal transferase.

Molecular characterization of the CmbR activator-binding site in the metC–cysK promoter region in Lactococcus lactis

Microbiology ◽  
2005 ◽  
Vol 151 (2) ◽  
pp. 439-446 ◽  
Author(s):  
Natasa Golic ◽  
Martijn Schliekelmann ◽  
María Fernández ◽  
Michiel Kleerebezem ◽  
Richard van Kranenburg
Keyword(s):  
Binding Site ◽  
Lactococcus Lactis ◽  
Promoter Region ◽  
Direct Repeat ◽  
Inverted Repeats ◽  
Sulphur Metabolism ◽  
Mobility Shift ◽  
Dna Fragment ◽  
Gel Mobility Shift

The metC–cysK operon involved in sulphur metabolism in Lactococcus lactis is positively regulated by the LysR-type protein CmbR. Transcription from the metC promoter is activated when concentrations of methionine and cysteine in the growth medium are low. The metC promoter region contains two direct and three inverted repeats. Deletion analysis indicated that direct repeat 2 (DR2) is required for activation of the metC promoter by CmbR. Gel mobility shift assays confirmed that CmbR binds to a 407 bp DNA fragment containing the metC promoter. This binding was stimulated by O-acetyl-l-serine. Competition experiments with deletion variants of the metC promoter showed that CmbR binding only occurred with fragments containing an intact DR2, confirming that DR2 is the CmbR binding site within the metC promoter.

scholarly journals Characterization of the maize Mutator transposable element MURA transposase as a DNA-binding protein.

1997 ◽  
Vol 17 (9) ◽  
pp. 5165-5175 ◽  
Author(s):  
M I Benito ◽  
V Walbot
Keyword(s):  
Dna Binding ◽  
Transposable Element ◽  
Binding Protein ◽  
Sequence Similarity ◽  
Functional Characterization ◽  
Dna Binding Protein ◽  
Amino Acid Sequence Similarity ◽  
Mobility Shift ◽  
Gel Mobility Shift

The autonomous MuDR element of the Mutator (Mu) transposable element family of maize encodes at least two proteins, MURA and MURB. Based on amino acid sequence similarity, previous studies have reported that MURA is likely to be a transposase. The functional characterization of MURA has been hindered by the instability of its cDNA, mudrA, in Escherichia coli. In this study, we report the first successful stabilization and expression of MURA in Saccharomyces cerevisiae. Gel mobility shift assays demonstrate that MURA is a DNA-binding protein that specifically binds to sequences within the highly conserved Mu element terminal inverted repeats (TIRs). DNase I and 1,10-phenanthroline-copper footprinting of MURA-Mu1 TIR complexes indicate that MURA binds to a conserved approximately 32-bp region in the TIR of Mu1. In addition, MURA can bind to the same region in the TIRs of all tested actively transposing Mu elements but binds poorly to the diverged Mu TIRs of inactive elements. Previous studies have reported a correlation between Mu transposon inactivation and methylation of the Mu element TIRs. Gel mobility shift assays demonstrate that MURA can interact differentially with unmethylated, hemimethylated, and homomethylated TIR substrates. The significance of MURA's interaction with the TIRs of Mu elements is discussed in the context of what is known about the regulation and mechanisms of Mutator activities in maize.

scholarly journals Functional and biochemical characterization of a recombinant Arabidopsis thaliana 3-deoxy-D-manno-octulosonate 8-phosphate synthase

2004 ◽  
Vol 381 (1) ◽  
pp. 185-193 ◽  
Author(s):  
Jing WU ◽  
Mayur A. PATEL ◽  
Appavu K. SUNDARAM ◽  
Ronald W. WOODARD
Keyword(s):  
Arabidopsis Thaliana ◽  
Molecular Mass ◽  
Biochemical Characterization ◽  
Dipicolinic Acid ◽  
Kinetic Studies ◽  
Maximum Activity ◽  
Reading Frame ◽  
Sequential Mechanism ◽  
Phosphate Synthase

An open reading frame, encoding for KDOPS (3-deoxy-D-manno-octulosonate 8-phosphate synthase), from Arabidopsis thaliana was cloned into a T7-driven expression vector. The protein was overexpressed in Escherichia coli and purified to homogeneity. Recombinant A. thaliana KDOPS, in solution, displays an apparent molecular mass of 76 kDa and a subunit molecular mass of 31.519 kDa. Unlike previously studied bacterial KDOPSs, which are tetrameric, A. thaliana KDOPS appears to be a dimer in solution. The optimum temperature of the enzyme is 65 °C and the optimum pH is 7.5, with a broad peak between pH 6.5 and 9.5 showing 90% of maximum activity. The enzyme cannot be inactivated by EDTA or dipicolinic acid treatment, nor it can be activated by a series of bivalent metal ions, suggesting that it is a non-metallo-enzyme, as opposed to the initial prediction that it would be a metallo-enzyme. Kinetic studies showed that the enzyme follows a sequential mechanism with Km=3.6 μM for phosphoenolpyruvate and 3.8 μM for D-arabinose 5-phosphate and kcat=5.9 s−1 at 37 °C. On the basis of the characterization of A. thaliana KDOPS and phylogenetic analysis, plant KDOPSs may represent a new, distinct class of KDOPSs.

scholarly journals Modulation of transcription and characterization of the promoter organization of the autotransporter adhesin heptosyltransferase and the autotransporter adhesin AIDA-I

Microbiology ◽  
2010 ◽  
Vol 156 (4) ◽  
pp. 1155-1166 ◽  
Author(s):  
Inga Benz ◽  
Tessa van Alen ◽  
Julia Bolte ◽  
Mirka E. Wörmann ◽  
M. Alexander Schmidt
Keyword(s):  
Protein Translocation ◽  
Growth Conditions ◽  
Limited Attention ◽  
Gram Negative Bacteria ◽  
Regulation Of Expression ◽  
Gene Encoding ◽  
E Coli ◽  
K 12 ◽  
Transcriptional Fusions

In Gram-negative bacteria, autotransporter proteins constitute the largest family of secreted proteins, and exhibit many different functions. In recent years, research has largely focused on mechanisms of autotransporter protein translocation, where several alternative models are still being discussed. In contrast, the biogenesis of only a few autotransporters has been studied and, likewise, regulation of expression has received only very limited attention. The glycosylated autotransporter adhesin involved in diffuse adherence (AIDA)-I system consists of the aah gene, encoding a specific autotransporter adhesin heptosyltransferase (AAH), and the aidA gene, encoding the autotransporter protein (AIDA-I). In this study, we investigated the promoter organization and transcription of these two genes using reporter plasmids carrying lacZ transcriptional fusions. The two genes, aah and aidA, are transcribed as a bicistronic message. However, aidA is additionally transcribed from its own promoter. There are two distinct start sites for each of the two genes. Interestingly, transcription of both genes is enhanced in hns and rfaH mutant backgrounds. Furthermore, we addressed the influence of environmental factors and different genetic backgrounds of Escherichia coli K-12 strains on transcription activity. We found that transcription varied considerably in different E. coli K-12 laboratory strains and under different growth conditions.

Characterization of a telomere-binding protein from Physarum polycephalum

1991 ◽  
Vol 11 (4) ◽  
pp. 2282-2290
Author(s):  
J S Coren ◽  
E M Epstein ◽  
V M Vogt
Keyword(s):  
Physarum Polycephalum ◽  
Nuclear Protein ◽  
Binding Activity ◽  
Binding Assays ◽  
Mobility Shift ◽  
Heat Stable ◽  
Dnase I Footprinting ◽  
Telomere Binding Protein ◽  
Gel Mobility Shift

We have partially purified a nuclear protein (PPT) from Physarum polycephalum that binds to the extrachromosomal ribosomal DNA telomeres of this acellular slime mold. Binding is specific for the (T2AG3)n telomere repeats, as evidenced by nitrocellulose filter binding assays, by gel mobility shift assays with both DNA fragments and double-stranded oligonucleotides, and by DNase I footprinting. PPT is remarkably heat stable, showing undiminished binding activity after incubation at 90 degrees C. It sediments at 1.2S, corresponding to a molecular weight of about 10,000 (for a globular protein), and its binding activity is undiminished by incubation with RNase, suggesting that it is not a ribonucleoprotein. We hypothesize that PPT plays a structural role in telomeres, perhaps preventing nucleolytic degradation or promoting telomere extension by a telomere-specific terminal transferase.

scholarly journals Regulatory Interactions among Adhesin Gene Systems of Uropathogenic Escherichia coli

2007 ◽  
Vol 76 (2) ◽  
pp. 771-780 ◽  
Author(s):  
Stina Lindberg ◽  
Yan Xia ◽  
Berit Sondén ◽  
Mikael Göransson ◽  
Jörg Hacker ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Regulatory Protein ◽  
Mobility Shift ◽  
E Coli ◽  
Fimbrial Adhesins ◽  
Gel Mobility Shift

ABSTRACT Uropathogenic Escherichia coli strain J96 carries multiple determinants for fimbrial adhesins. The regulatory protein PapB of P fimbriae has previously been implicated in potential coregulatory events. The focB gene of the F1C fimbria determinant is highly homologous to papB; the translated sequences share 81% identity. In this study we investigated the role of PapB and FocB in regulation of the F1C fimbriae. By using gel mobility shift assays, we showed that FocB binds to sequences in both the pap and foc operons in a somewhat different manner than PapB. The results of both in vitro cross-linking and in vivo oligomerization tests indicated that FocB could function in an oligomeric fashion. Furthermore, our results suggest that PapB and FocB can form heterodimers and that these complexes can repress expression of the foc operon. The effect of FocB on expression of type 1 fimbriae was also tested. Taken together, the results that we present expand our knowledge about a regulatory network for different adhesin gene systems in uropathogenic E. coli and suggest a hierarchy for expression of the fimbrial adhesins.

scholarly journals Salmonella enteritidis: AmpC Plasmid-Mediated Inducible β-Lactamase (DHA-1) with anampR Gene from Morganella morganii

1998 ◽  
Vol 42 (9) ◽  
pp. 2352-2358 ◽  
Author(s):  
Guilene Barnaud ◽  
Guillaume Arlet ◽  
Charlotte Verdet ◽  
Olivier Gaillot ◽  
Philippe H. Lagrange ◽  
...  
Keyword(s):  
Dna Sequence ◽  
Salmonella Enteritidis ◽  
Diffusion Method ◽  
Morganella Morganii ◽  
Mobility Shift ◽  
Reading Frame ◽  
E Coli ◽  
Amino Terminal ◽  
Mobility Shift Assay ◽  
Gel Mobility Shift

ABSTRACT DHA-1, a plasmid-mediated cephalosporinase from a single clinicalSalmonella enteritidis isolate, conferred resistance to oxyimino-cephalosporins (cefotaxime and ceftazidime) and cephamycins (cefoxitin and moxalactam), and this resistance was transferable toEscherichia coli HB101. An antagonism was observed between cefoxitin and aztreonam by the diffusion method. Transformation of the transconjugant E. coli strain with plasmid pNH5 carrying the ampD gene (whose product decreases the level of expression of ampC) resulted in an eightfold decrease in the MIC of cefoxitin. A clone with the same AmpC susceptibility pattern with antagonism was obtained, clone E. coliJM101(pSAL2-ind), and its nucleotide sequence was determined. It contained an open reading frame with 98.7% DNA sequence identity with the ampC gene of Morganella morganii. DNA sequence analysis also identified a gene upstream of ampCwhose sequence was 97% identical to the partial sequence of the ampR gene (435 bp) from M. morganii. The gene encoded a protein with an amino-terminal DNA-binding domain typical of transcriptional activators of the LysR family. Moreover, the intercistronic region between the ampC andampR genes was 98% identical to the corresponding region from M. morganii DNA. AmpR was shown to be functional by enzyme induction and a gel mobility-shift assay. An ampGgene was also detected in a Southern blot of DNA from the S. enteritidis isolate. These findings suggest that this inducible plasmid-mediated AmpC type β-lactamase, DHA-1, probably originated from M. morganii.

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