SoxRS-mediated regulation of chemotrophic sulfur oxidation in Paracoccus pantotrophus

Microbiology ◽  
2005 ◽  
Vol 151 (5) ◽  
pp. 1707-1716 ◽  
Author(s):  
Dagmar Rother ◽  
Grazyna Orawski ◽  
Frank Bardischewsky ◽  
Cornelius G. Friedrich
Keyword(s):  
Sequence Analysis ◽  
Sulfur Oxidation ◽  
Growth Conditions ◽  
Kanamycin Resistance ◽  
Cysteine Residue ◽  
Mobility Shift ◽  
C Terminus ◽  
Intergenic Regions ◽  
Paracoccus Pantotrophus ◽  
Gel Mobility Shift

Paracoccus pantotrophus GB17 requires thiosulfate for induction of the sulfur-oxidizing (Sox) enzyme system. The soxRS genes are divergently oriented to the soxVWXYZA–H genes. soxR predicts a transcriptional regulator of the ArsR family and soxS a periplasmic thioredoxin. The homogenote mutant GBΩS carrying a disruption of soxS by the Ω-kanamycin-resistance-encoding interposon expressed a low thiosulfate-oxidizing activity under heterotrophic and mixotrophic growth conditions. This activity was repressed by complementation with soxR, suggesting that SoxR acts as a repressor and SoxS is essential for full expression. Sequence analysis uncovered operator characteristics in the intergenic regions soxS–soxV and soxW–soxX. In each region a transcription start site was identified by primer extension analysis. Both regions were cloned into the vector pRI1 and transferred to P. pantotrophus. Strains harbouring pRI1 with soxS–soxV or soxW–soxX expressed the sox genes under heterotrophic conditions at a low rate, indicating repressor titration. Sequence analysis of SoxR suggested a helix–turn–helix (HTH) motif at position 87–108 and uncovered an invariant Cys-80 and a cysteine residue at the C-terminus. SoxR was overproduced in Escherichia coli with an N-terminal His6-tag and purified to near homogeneity. Electrophoretic gel mobility shift assays with SoxR retarded the soxS–soxV region as a single band while the soxW–soxX region revealed at least two protein–DNA complexes. These data demonstrated binding of SoxR to the relevant DNA. This is believed to be the first report of regulation of chemotrophic sulfur oxidation at the molecular level.

Influence of homologous phasins (PhaP) on PHA accumulation and regulation of their expression by the transcriptional repressor PhaR in Ralstonia eutropha H16

Microbiology ◽  
2005 ◽  
Vol 151 (3) ◽  
pp. 825-833 ◽  
Author(s):  
Markus Pötter ◽  
Helena Müller ◽  
Alexander Steinbüchel
Keyword(s):  
Ralstonia Eutropha ◽  
Current Model ◽  
Transcriptional Repressor ◽  
Start Codon ◽  
Wild Type ◽  
Mobility Shift ◽  
Intergenic Regions ◽  
Gel Mobility Shift ◽  
Dnasei Footprinting ◽  
Similar Growth

Phasins play an important role in the formation of poly(3-hydroxybutyrate) [poly(3HB)] granules and affect their size. Recently, three homologues of the phasin protein PhaP1 were identified in Ralstonia eutropha strain H16. The functions of PhaP2, PhaP3 and PhaP4 were examined by analysis of R. eutropha H16 deletion strains (ΔphaP1, ΔphaP2, ΔphaP3, ΔphaP4, ΔphaP12, ΔphaP123 and ΔphaP1234). When cells were grown under conditions permissive for poly(3HB) accumulation, the wild-type strain and all single-phasin negative mutants (ΔphaP2, ΔphaP3 and ΔphaP4), with the exception of ΔphaP1, showed similar growth and poly(3HB) accumulation behaviour, and also the size and number of the granules were identical. The single ΔphaP1 mutant and the ΔphaP12, ΔphaP123 and ΔphaP1234 mutants showed an almost identical growth behaviour; however, they accumulated poly(3HB) at a significantly lower level than wild-type and the single ΔphaP2, ΔphaP3 or ΔphaP4 mutants. Gel-mobility-shift assays and DNaseI footprinting experiments demonstrated the capability of the transcriptional repressor PhaR to bind to a DNA region +36 to +46 bp downstream of the phaP3 start codon. The protected sequence exhibited high similarity to the binding sites of PhaR upstream of phaP1, which were identified recently. In contrast, PhaR did not bind to the upstream or intergenic regions of phaP2 and phaP4, thus indicating that the expression of these two phasins is regulated in a different way. Our current model for the regulation of phasins in R. eutropha strain H16 was extended and confirmed.

scholarly journals 3′-immature tRNATrp is required for ribosome inactivation by gelonin,a plant RNA N-glycosidase

1995 ◽  
Vol 310 (1) ◽  
pp. 249-253 ◽  
Author(s):  
M Brigotti ◽  
D Carnicelli ◽  
P Alvergna ◽  
A Pallanca ◽  
R Lorenzetti ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Liver Extract ◽  
Ribonuclease H ◽  
Ribosome Inactivating Protein ◽  
Mobility Shift ◽  
Glycosidase Activity ◽  
Ribosome Inactivation ◽  
Gel Mobility Shift ◽  
High Level ◽  
Good Acceptor

Inactivation of ribosomes by gelonin, a ribosome-inactivating protein with RNA N-glycosidase activity on 28 S rRNA, requires macromolecular cofactors present in post-ribosomal supernatants. One of these cofactors has been purified from a rat liver extract and identified as an RNA about 70 nt long which in sequence analysis shows a high level of similarity with mammalian (bovine) tRNA(Trp). The pattern of the sequencing gel is consistent with the co-existence in the preparation of two 3′-immature tRNA(Trp) species, missing only A75, or both A75 and C74. In the presence of ATP, CTP and tRNA nucleotidyltransferase, the gelonin-stimulating RNA is a good acceptor of tryptophan. An oligodeoxynucleotide complementary to positions 55 to 72 of mammalian (bovine) tRNA(Trp) hybridizes with the gelonin-stimulating RNA as demonstrated by gel mobility shift and ribonuclease H digestion. The oligodeoxynucleotide-directed ribonuclease H treatment also abolishes the gelonin-promoting activity of crude preparations of RNA, giving strong evidence that the only active RNA is a tRNA(Trp)-like molecule.

scholarly journals runt Homology Domain Transcription Factors (Runx, Cbfa, and AML) Mediate Repression of the Bone Sialoprotein Promoter: Evidence for Promoter Context-Dependent Activity of Cbfa Proteins

2001 ◽  
Vol 21 (8) ◽  
pp. 2891-2905 ◽  
Author(s):  
Amjad Javed ◽  
George L. Barnes ◽  
B. O. Jasanya ◽  
Janet L. Stein ◽  
Louis Gerstenfeld ◽  
...  
Keyword(s):  
Amino Acids ◽  
Bone Development ◽  
Bone Sialoprotein ◽  
Mobility Shift ◽  
Suppressor Activity ◽  
Nuclear Extracts ◽  
C Terminus ◽  
Mineralized Tissues ◽  
Functional Involvement ◽  
Gel Mobility Shift

ABSTRACT Expression of the bone sialoprotein (BSP) gene, a marker of bone formation, is largely restricted to cells in mineralized tissues. Recent studies have shown that the Cbfa1 (also known as Runx2, AML-3, and PEBP2αA) transcription factor supports commitment and differentiation of progenitor cells to hypertrophic chondrocytes and osteoblasts. This study addresses the functional involvement of Cbfa sites in expression of the Gallus BSP gene. Gel mobility shift analyses with nuclear extracts from ROS 17/2.8 osteoblastic cells revealed that multiple Cbfa consensus sequences are functional Cbfa DNA binding sites. Responsiveness of the 1.2-kb Gallus BSP promoter to Cbfa factors Cbfa1, Cbfa2, and Cbfa3 was assayed in osseous and nonosseous cells. Each of the Cbfa factors mediated repression of the wild-type BSP promoter, in contrast to their well known activation of various hematopoietic and skeletal phenotypic genes. Suppression of BSP by Cbfa factors was not observed in BSP promoters in which Cbfa sites were deleted or mutated. Expression of the endogenous BSP gene inGallus osteoblasts was similarly downregulated by forced expression of Cbfa factors. Our data indicate that Cbfa repression of the BSP promoter does not involve the transducin-like enhancer (TLE) proteins. Neither coexpression of TLE1 or TLE2 nor the absence of the TLE interaction motif of Cbfa1 (amino acids 501 to 513) influenced repressor activity. However, removal of the C terminus of Cbfa1 (amino acids 362 to 513) relieved suppression of the BSP promoter. Our results, together with the evolutionary conservation of the seven Cbfa sites in the Gallus and human BSP promoters, suggest that suppressor activity by Cbfa is of significant physiologic consequence and may contribute to spatiotemporal expression of BSP during bone development.

scholarly journals Saccharomyces cerevisiae HSP70 heat shock elements are functionally distinct.

1993 ◽  
Vol 13 (9) ◽  
pp. 5637-5646 ◽  
Author(s):  
M R Young ◽  
E A Craig
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Point Mutations ◽  
Growth Conditions ◽  
Mobility Shift ◽  
Basal Transcription ◽  
Basal Activity ◽  
Flanking Sequences ◽  
Gel Mobility Shift ◽  
Ability To Drive

The Saccharomyces cerevisiae HSP70 gene SSA1 has multiple heat shock elements (HSEs). To determine the significance of each of these sequences for expression of SSA1, we analyzed expression from a set of promoters containing point mutations in each of the HSEs, individually and in pairwise combinations. Of the three HSE-like sequences, two (HSE2 and HSE3) were active promoter elements; only one, HSE2, was active under basal growth conditions. Either HSE2 or HSE3 alone was able to drive SSA1 transcription at near-normal rates after heat shock. Both HSE2 and HSE3 were capable of driving basal transcription when placed in the context of the CYC1 promoter. Previous analysis had identified an upstream repressing sequence overlapping HSE2 that repressed basal transcription driven by HSE2. Our analysis showed that basal transcription driven by HSE3 was repressed both by the distant upstream repressing sequence and by closer flanking sequences. The ability to drive basal transcription is not inherent in all natural HSEs, since the HSEs from the heat-inducible SSA3 and SSA4 genes showed no basal activity when placed in the CYC1 vector. Gel mobility shift experiments showed that the same population of heat shock transcription factor molecules bound to HSEs capable of driving basal activity and to HSEs having very low or undetectable basal activity.

NF1 transcriptional factor(s) is required for basal promoter activation of the human intestinal NaPi-IIb cotransporter gene

2005 ◽  
Vol 288 (2) ◽  
pp. G175-G181 ◽  
Author(s):  
Hua Xu ◽  
Jennifer K. Uno ◽  
Michael Inouye ◽  
James F. Collins ◽  
Fayez K. Ghishan
Keyword(s):  
Gc Content ◽  
Gene Promoter ◽  
Normal Growth ◽  
Protein S ◽  
Growth Conditions ◽  
Transcriptional Factor ◽  
Minimal Promoter ◽  
Mobility Shift ◽  
Nuclear Factor 1 ◽  
Gel Mobility Shift

The human intestinal type IIb Na+-Picotransporter (hNaPi-IIb) gene promoter lacks a TATA box and has a high GC content in the 5′-flanking region. To understand the mechanism of hNaPi-IIb gene transcription, the current study was performed to characterize the minimal promoter region and transcriptional factor(s) necessary to activate gene expression in human intestinal cells (Caco-2). With the use of progressively shorter promoter constructs, a minimal promoter extending from bp −58 to +15 was identified and shown to direct high levels of hNaPi-IIb cotransporter expression in Caco-2 cells. Gel mobility shift assays (GMSAs) indicated that two regions could be bound by nuclear proteins from Caco-2 cells: region A at bp −26/−23 and region B at bp −44/−35. The introduction of mutations in region A abolished promoter activity, whereas mutations in region B had no effect. Deletion mutants of the same regions showed identical results. Furthermore, DNase I footprinting experiments confirmed the observation made by GMSAs. Additional studies, which used a specific nuclear factor 1 (NF1) antiserum, demonstrated that NF1 protein(s) binds to the minimal promoter at region A. These results indicated that the NF1 protein(s) is required to activate the basal transcription of hNaPi-IIb gene under normal growth conditions. This study has thus identified a new target gene in the small intestinal epithelium that is directly regulated by NF1 transcriptional factor(s).

scholarly journals The DNA Binding Domains of P1 ParB and the Architecture of the P1 Plasmid Partition Complex

2001 ◽  
Vol 276 (15) ◽  
pp. 12385-12394 ◽  
Author(s):  
Jennifer A. Surtees ◽  
Barbara E. Funnell
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Integration Host Factor ◽  
Mobility Shift ◽  
Dna Binding Domains ◽  
High Affinity ◽  
Binding Domains ◽  
Dna Binding Activity ◽  
C Terminus ◽  
Gel Mobility Shift

Stable maintenance of P1 plasmids inEscherichia coliis mediated by a high affinity nucleoprotein complex called the partition complex, which consists of ParB and theE. coliintegration host factor (IHF) bound specifically to the P1parSsite. IHF strongly stimulates ParB binding toparS, and the minimal partition complex contains a single dimer of ParB. To examine the architecture of the partition complex, we have investigated the DNA binding activity of various ParB fragments. Gel mobility shift and DNase I protection assays showed that the first 141 residues of ParB are dispensable for the formation of the minimal, high affinity partition complex. A fragment missing only the last 16 amino acids of ParB bound specifically toparS, but binding was weak and was no longer stimulated by IHF. The ability of IHF to stimulate ParB binding toparScorrelated with the ability of ParB to dimerize via its C terminus. Using full and partialparSsites, we show that two regions of ParB, one in the center and the other near the C terminus of the protein, interact with distinct sequences withinparS. Based on these data, we have proposed a model of how the ParB dimer bindsparSto form the minimal partition complex.

scholarly journals SoxV transfers electrons to the periplasm of Paracoccus pantotrophus – an essential reaction for chemotrophic sulfur oxidation

Microbiology ◽  
2006 ◽  
Vol 152 (2) ◽  
pp. 465-472 ◽  
Author(s):  
Frank Bardischewsky ◽  
Jörg Fischer ◽  
Bettina Höller ◽  
Cornelius G. Friedrich
Keyword(s):  
Dna Sequences ◽  
System Analysis ◽  
Sulfur Oxidation ◽  
Kanamycin Resistance ◽  
Redox Partner ◽  
Functional Homology ◽  
Cell Extracts ◽  
Genes Encoding ◽  
Paracoccus Pantotrophus

The soxVW genes are located upstream of the sox gene cluster encoding the sulfur-oxidizing ability of Paracoccus pantotrophus. SoxV is highly homologous to CcdA, which is involved in cytochrome c maturation of P. pantotrophus. SoxV was shown to function in reduction of the periplasmic SoxW, which shows a CysXaaXaaCys motif characteristic for thioredoxins. From strain GBΩV, which carries an Ω-kanamycin-resistance-encoding interposon in soxV, and complementation analysis it was evident that SoxV but not the periplasmic SoxW was essential for lithoautotrophic growth of P. pantotrophus with thiosulfate. However, the thiosulfate-oxidizing activities of cell extracts from the wild-type and from strain GBΩV were similar, demonstrating that the low thiosulfate-oxidizing activity of strain GBΩV in vivo was not due to a defect in biosynthesis or maturation of proteins of the Sox system and suggesting that SoxV is part of a regulatory or catalytic system of the Sox system. Analysis of DNA sequences available from different organisms harbouring a Sox system revealed that soxVW genes are exclusively present in sox operons harbouring the soxCD genes, encoding sulfur dehydrogenase, suggesting that SoxCD might be a redox partner of SoxV. No complementation of the ccdA mutant P. pantotrophus TP43 defective in cytochrome c maturation was achieved by expression of soxV in trans, demonstrating that the high identity of SoxV and CcdA does not correspond to functional homology.

scholarly journals Saccharomyces cerevisiae HSP70 heat shock elements are functionally distinct

1993 ◽  
Vol 13 (9) ◽  
pp. 5637-5646
Author(s):  
M R Young ◽  
E A Craig
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Point Mutations ◽  
Growth Conditions ◽  
Mobility Shift ◽  
Basal Transcription ◽  
Basal Activity ◽  
Flanking Sequences ◽  
Gel Mobility Shift ◽  
Ability To Drive

The Saccharomyces cerevisiae HSP70 gene SSA1 has multiple heat shock elements (HSEs). To determine the significance of each of these sequences for expression of SSA1, we analyzed expression from a set of promoters containing point mutations in each of the HSEs, individually and in pairwise combinations. Of the three HSE-like sequences, two (HSE2 and HSE3) were active promoter elements; only one, HSE2, was active under basal growth conditions. Either HSE2 or HSE3 alone was able to drive SSA1 transcription at near-normal rates after heat shock. Both HSE2 and HSE3 were capable of driving basal transcription when placed in the context of the CYC1 promoter. Previous analysis had identified an upstream repressing sequence overlapping HSE2 that repressed basal transcription driven by HSE2. Our analysis showed that basal transcription driven by HSE3 was repressed both by the distant upstream repressing sequence and by closer flanking sequences. The ability to drive basal transcription is not inherent in all natural HSEs, since the HSEs from the heat-inducible SSA3 and SSA4 genes showed no basal activity when placed in the CYC1 vector. Gel mobility shift experiments showed that the same population of heat shock transcription factor molecules bound to HSEs capable of driving basal activity and to HSEs having very low or undetectable basal activity.

Snapshot blotting: The transfer of nucleic acids and nucleoprotein complexes from electrophoresis gels to EM grids

1992 ◽  
Vol 50 (1) ◽  
pp. 762-763
Author(s):  
Stephen D. Jett
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Nucleic Acid Binding ◽  
Mobility Shift ◽  
Carbon Coated ◽  
Nucleoprotein Complexes ◽  
Mobility Shift Assay ◽  
Protein Nucleic Acid ◽  
Gel Mobility Shift ◽  
Nucleic Acid Interactions

The electrophoresis gel mobility shift assay is a popular method for the study of protein-nucleic acid interactions. The binding of proteins to DNA is characterized by a reduction in the electrophoretic mobility of the nucleic acid. Binding affinity, stoichiometry, and kinetics can be obtained from such assays; however, it is often desirable to image the various species in the gel bands using TEM. Present methods for isolation of nucleoproteins from gel bands are inefficient and often destroy the native structure of the complexes. We have developed a technique, called “snapshot blotting,” by which nucleic acids and nucleoprotein complexes in electrophoresis gels can be electrophoretically transferred directly onto carbon-coated grids for TEM imaging.

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