scholarly journals Independent Regulation of MucD, an HtrA-Like Protease in Pseudomonas aeruginosa, and the Role of Its Proteolytic Motif in Alginate Gene Regulation

2006 ◽  
Vol 188 (8) ◽  
pp. 3134-3137 ◽  
Author(s):  
Lynn F. Wood ◽  
Dennis E. Ohman
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Gene Regulation ◽  
Transcriptional Start Site ◽  
Single Copy ◽  
Start Site ◽  
Temperature Resistance

ABSTRACT Expression of mucD, encoding a homologue of the HtrA(DegP) family of endoserine proteases, was investigated in Pseudomonas aeruginosa. Expressed from the algT-mucABCD operon, MucD was detected in mucoid (FRD1) and nonmucoid (PAO1) parental strains and also when polar insertions were placed upstream in algT or mucB. A transcriptional start site for a mucD promoter (PmucD) was mapped within mucC. Expression of single-copy mucD217, encoding MucD altered in the protease motif (S217A), was defective in temperature resistance and alginate gene regulation.

scholarly journals Effects of Iron and Temperature on Expression of the Pseudomonas aeruginosa tolQRA Genes: Role of the Ferric Uptake Regulator

1998 ◽  
Vol 180 (11) ◽  
pp. 2836-2841 ◽  
Author(s):  
Eric R. Lafontaine ◽  
Pamela A. Sokol
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Transcriptional Start Site ◽  
Regulatory Gene ◽  
Iron Regulation ◽  
Northern Hybridization ◽  
Ferric Uptake Regulator ◽  
Start Site ◽  
Promoter Regions

ABSTRACT The tolQRA genes have been recently identified inPseudomonas aeruginosa PAO. In this study, we examined the effect of iron and temperature on tolQRA expression. A promoterless lacZ gene was introduced downstream of plasmid-encoded tolQ and tolA, and expression was monitored by measuring β-galactosidase activity of cultures. Addition of 25 μM FeCl3 to the culture medium reduced tolQRA expression by 50 to 60% in PAO but by only 25% in the fur mutant PAO A4. Northern hybridization analysis revealed that iron regulation occurs at the level of transcription and involves the P. aeruginosaferric uptake regulator (Fur). Primer extension analysis was used to identify the proposed transcriptional start site oftolA. Although a putative Fur box was identified 20 bp upstream of the proposed start site, purified Fur did not bind to thetolA or tolQR promoter regions in an in vitro gel retardation assay. Therefore, iron regulation of thetol genes appears to involve an intermediate regulatory gene. Expression of tolQR andtolA was optimal at 37°C and was reduced by 40 to 50% when cultures were grown at either 42 or 25°C. Growth in high-iron medium at 25°C further reduced tolQR andtolA expression.

scholarly journals The chromatin remodeler Ino80 mediates RNAPII pausing site determination

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Youngseo Cheon ◽  
Sungwook Han ◽  
Taemook Kim ◽  
Daehee Hwang ◽  
Daeyoup Lee
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Start Site ◽  
Regulation Of Gene Expression ◽  
Embryonic Stem ◽  
Start Site ◽  
Chromatin Remodeler ◽  
Close Relationship ◽  
Early Transcription ◽  
Different Characteristics

Abstract Background Promoter-proximal pausing of RNA polymerase II (RNAPII) is a critical step for the precise regulation of gene expression. Despite the apparent close relationship between promoter-proximal pausing and nucleosome, the role of chromatin remodeler governing this step has mainly remained elusive. Results Here, we report highly confined RNAPII enrichments downstream of the transcriptional start site in Saccharomyces cerevisiae using PRO-seq experiments. This non-uniform distribution of RNAPII exhibits both similar and different characteristics with promoter-proximal pausing in Schizosaccharomyces pombe and metazoans. Interestingly, we find that Ino80p knockdown causes a significant upstream transition of promoter-proximal RNAPII for a subset of genes, relocating RNAPII from the main pausing site to the alternative pausing site. The proper positioning of RNAPII is largely dependent on nucleosome context. We reveal that the alternative pausing site is closely associated with the + 1 nucleosome, and nucleosome architecture around the main pausing site of these genes is highly phased. In addition, Ino80p knockdown results in an increase in fuzziness and a decrease in stability of the + 1 nucleosome. Furthermore, the loss of INO80 also leads to the shift of promoter-proximal RNAPII toward the alternative pausing site in mouse embryonic stem cells. Conclusions Based on our collective results, we hypothesize that the highly conserved chromatin remodeler Ino80p is essential in establishing intact RNAPII pausing during early transcription elongation in various organisms, from budding yeast to mouse.

scholarly journals Binding of Pseudomonas aeruginosa AlgZ to Sites Upstream of the algZ Promoter Leads to Repression of Transcription

2005 ◽  
Vol 187 (13) ◽  
pp. 4430-4443 ◽  
Author(s):  
Deborah M. Ramsey ◽  
Patricia J. Baynham ◽  
Daniel J. Wozniak
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Binding Sites ◽  
Transcriptional Control ◽  
Specific Binding ◽  
Transcriptional Start Site ◽  
Opportunistic Pathogen ◽  
Single Copy ◽  
Amino Acid Identity ◽  
Mobility Shift ◽  
Base Pairs

ABSTRACT Mucoid variants of the opportunistic pathogen Pseudomonas aeruginosa produce the exopolysaccharide alginate and colonize the respiratory tracts of cystic fibrosis patients. The genes encoding the alginate biosynthetic enzymes are clustered in a single operon, which is under tight transcriptional control. One essential activator of the alginate operon is AlgZ, a proposed ribbon-helix-helix DNA binding protein that shares 30% amino acid identity with the Mnt repressor of Salmonella enterica serovar Typhimurium bacteriophage P22. In the current study, we examined the role of AlgZ as an autoregulator. Using single-copy algZ-lacZ transcription fusions, an increase in algZ transcription was observed in an algZ mutant compared to the isogenic wild-type strain, suggesting that AlgZ may have an additional role as a repressor. To identify the AlgZ binding site, overlapping regions upstream of algZ were incubated with AlgZ and analyzed by electrophoretic mobility shift assays. Specific binding activity was localized to a region spanning from 66 to 185 base pairs upstream of the algZ transcriptional start site. Two AlgZ binding sites were defined using copper-phenanthroline footprinting and deletion analyses, with one site centered at 93 base pairs and the other centered at 161 base pairs upstream of the algZ promoter. Deletion of both binding sites resulted in the loss of AlgZ binding. These results indicate that AlgZ represses algZ transcription, and this activity is mediated by multiple AlgZ-DNA interactions.

scholarly journals The Pseudomonas aeruginosa phosphodiesterase gene nbdA is transcriptionally regulated by RpoS and AmrZ

2021 ◽  
Author(s):  
Katrin Gerbracht ◽  
Susanne Zehner ◽  
Nicole Frankenberg-Dinkel
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Transcriptional Regulation ◽  
Promoter Region ◽  
Transcriptional Start Site ◽  
Guanosine Monophosphate ◽  
Enzymatic Function ◽  
Nitrite Reductase Gene ◽  
Biofilm Dispersal ◽  
The Impact

Pseudomonas aeruginosa is an opportunistic pathogen causing serious infections in immune compromised persons. These infections are difficult to erase with antibiotics, due to the formation of biofilms. The biofilm lifecycle is regulated by the second messenger molecule c-di-GMP (bis-3,5-cyclic di-guanosine monophosphate). P. aeruginosa encodes 40 genes for enzymes presumably involved in the biosynthesis and degradation of c-di-GMP. A tight regulation of expression, subcellular localized function and protein interactions control the activity of these enzymes. In this work we elucidated the transcriptional regulation of the gene encoding the membrane-bound phosphodiesterase NbdA. We previously reported a transcriptional and posttranslational role of nitric oxide (NO) on nbdA and its involvement in biofilm dispersal. NO is released from macrophages during infections but can also be produced by P. aeruginosa itself during anaerobic denitrification. Recently however, contradictory results about the role of NbdA within NO-induced biofilm dispersal were published. Therefore, the transcriptional regulation of nbdA was reevaluated to obtain insights into this discrepancy. Determination of the transcriptional start site of nbdA by 5'-RACE and subsequent identification of the promoter region revealed a shortened open reading frame (ORF) in contrast to the annotated one. In addition, putative binding sites for RpoS and AmrZ were discovered in the newly defined promoter region. Employing chromosomally integrated transcriptional lacZ reporter gene fusions demonstrated a RpoS-dependent activation and AmrZ repression of nbdA transcription. In order to investigate the impact of NO on nbdA transcription, conditions mimicking exogenous and endogenous NO were applied. While neither exogenous nor endogenous NO had an influence on nbdA promoter activity, deletion of the nitrite reductase gene nirS strongly increased nbdA transcription independently of its enzymatic activity during denitrification. The latter supports a role of NirS in P. aeruginosa apart from its enzymatic function.

scholarly journals Expanding the repertoire of glucocorticoid receptor target genes by engineering genomic response elements

2018 ◽  
Author(s):  
Verena Thormann ◽  
Laura V. Glaser ◽  
Maika C. Rothkegel ◽  
Marina Borschiwer ◽  
Melissa Bothe ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Glucocorticoid Receptor ◽  
Binding Site ◽  
Genome Engineering ◽  
Target Genes ◽  
Transcriptional Start Site ◽  
Chromatin Accessibility ◽  
Start Site ◽  
Response Elements ◽  
Genomic Response

The glucocorticoid receptor (GR), a hormone-activated transcription factor, binds to a myriad of genomic binding sites yet seems to regulate a much smaller number of genes. Genome-wide analysis of GR binding and gene regulation has shown that the likelihood of GR-dependent regulation increases with decreased distance of its binding to the transcriptional start site of a gene. To test if we can adopt this knowledge to expand the repertoire of GR target genes, we used homology directed repair (HDR)-mediated genome editing to add a single GR binding site directly upstream of the transcriptional start site of four genes. To our surprise, we found that the addition of a single GR binding site can be enough to convert a gene into a GR target. The gain of GR-dependent regulation was observed for two out of four genes analyzed and coincided with acquired GR binding at the introduced binding site. However, the gene-specific gain of GR-dependent regulation could not be explained by obvious differences in chromatin accessibility between converted genes and their non-converted counterparts. Further, by introducing GR binding sequences with different nucleotide compositions, we show that activation can be facilitated by distinct sequences without obvious differences in activity between the GR binding sequence variants we tested. The approach to use genome engineering to build genomic response elements facilitates the generation of cell lines with tailored repertoires of GR-responsive genes and a framework to test and refine our understanding of the cis-regulatory logic of gene regulation by testing if engineered response elements behave as predicted.

scholarly journals Regulation of the Carnitine Pathway inEscherichia coli: Investigation of the cai-fixDivergent Promoter Region

1998 ◽  
Vol 180 (10) ◽  
pp. 2599-2608 ◽  
Author(s):  
Anne Buchet ◽  
Knut Eichler ◽  
Marie-Andrée Mandrand-Berthelot
Keyword(s):  
Transcriptional Start Site ◽  
Regulatory Protein ◽  
Regulatory Region ◽  
Site Directed Mutagenesis ◽  
Anaerobic Growth ◽  
Start Site ◽  
Direct Role ◽  
Dnase I Footprinting ◽  
Carnitine Metabolism

ABSTRACT The divergent structural operons caiTABCDE andfixABCX of Escherichia coli are required for anaerobic carnitine metabolism. Transcriptional monocopylacZ fusion studies showed that both operons are coexpressed during anaerobic growth in the presence of carnitine, respond to common environmental stimuli (like glucose and nitrate), and are modulated positively by the same general regulators, CRP and FNR, and negatively by H-NS. Overproduction of the CaiF specific regulatory protein mediating the carnitine signal restored induction in anfnr mutant, corresponding to its role as the primary target for anaerobiosis. Transcript analysis identified two divergent transcription start points initiating 289 bp apart. DNase I footprinting revealed three sites with various affinities for the binding of the cAMP-CRP complex inside this regulatory region. Site-directed mutagenesis experiments indicated that previously reported perfect CRP motif 1, centered at −41.5 of the caitranscriptional start site, plays a direct role in the solecai activation. In contrast, mutation in CRP site 2, positioned at −69.5 of the fix promoter, caused only a threefold reduction in fix expression. Thus, the role of the third CRP site, located at −126.5 of fix, might be to reinforce the action of site 2. A critical 50-bp cis-acting sequence overlapping the fix mRNA start site was found, by deletion analysis, to be necessary for cai transcription. This region is thought to be involved in transduction of the signal mediated by the CaiF regulator.

scholarly journals Sequence and transcriptional start site of the Pseudomonas aeruginosa outer membrane porin protein F gene.

1988 ◽  
Vol 170 (1) ◽  
pp. 155-162 ◽  
Author(s):  
M Duchêne ◽  
A Schweizer ◽  
F Lottspeich ◽  
G Krauss ◽  
M Marget ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Transcriptional Start Site ◽  
Start Site ◽  
F Gene ◽  
Porin Protein ◽  
Outer Membrane Porin ◽  
Protein F

scholarly journals Role of Nucleoid-Associated Proteins Hha and H-NS in Expression of Salmonella enterica Activators HilD, HilC, and RtsA Required for Cell Invasion

2007 ◽  
Vol 189 (19) ◽  
pp. 6882-6890 ◽  
Author(s):  
Igor N. Olekhnovich ◽  
Robert J. Kadner
Keyword(s):  
Salmonella Enterica ◽  
Transcriptional Start Site ◽  
Single Copy ◽  
Negative Control ◽  
Transcriptional Activators ◽  
Mobility Shift ◽  
Dna Binding Site ◽  
Associated Proteins ◽  
Complex Circuits

ABSTRACT The coordinate expression of Salmonella enterica invasion genes on Salmonella pathogenicity island 1 is under the control of the complex circuits of regulation that involve the AraC/XylS family transcriptional activators HilD, HilC, and RtsA and nucleoid-associated proteins. Single-copy transcription fusions were used to assess the effects of nucleoid-associated proteins Hha and H-NS on hilD, hilC, and rtsA expression. The data show that all three genes, hilD, hilC, and rtsA, were repressed by H-NS and/or Hha. The repression of rtsA was the highest among tested genes. The level of rtsA-lac was equally elevated in hns and hha mutants and was further enhanced in the hns hha double mutant under low-osmolarity conditions. Electrophoretic mobility shift experiments showed that H-NS and Hha directly bind to the rtsA promoter. In addition to the negative control that was exerted by H-NS/Hha under low-osmolarity conditions, the homologous virulence activators HilD, HilC, and RtsA (Hil activators) induced rtsA-lac expression in a high-salt medium. A DNase footprinting assay of the rtsA promoter revealed one common DNA-binding site for all three Hil activators centered at position −54 relative to the transcriptional start site. In the absence of Hha and H-NS, however, osmoregulation of the rtsA promoter was lost, and Hil activators were not required for rtsA transcription. These results taken together suggest that the HilD, HilC, and RtsA proteins induce the transcription of the rtsA promoter by counteracting H-NS/Hha-mediated repression.

scholarly journals Functional Studies of CCAAT/Enhancer Binding Protein Site Located Downstream of the Transcriptional Start Site

2017 ◽  
Vol 10 ◽  
pp. 117955571769455 ◽  
Author(s):  
Yujie Liu ◽  
Michael R Nonnemacher ◽  
Aikaterini Alexaki ◽  
Vanessa Pirrone ◽  
Anupam Banerjee ◽  
...  
Keyword(s):  
Binding Protein ◽  
Transcriptional Start Site ◽  
Replication Capacity ◽  
Start Site ◽  
Transcriptional Initiation ◽  
Functional Studies ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein ◽  
Hiv 1

Previous studies have identified a CCAAT/enhancer binding protein (C/EBP) site located downstream of the transcriptional start site (DS3). The role of the DS3 element with respect to HIV-1 transactivation by Tat and viral replication has not been characterized. We have demonstrated that DS3 was a functional C/EBPβ binding site and mutation of this site to the C/EBP knockout DS3-9C variant showed lower HIV-1 long terminal repeat (LTR) transactivation by C/EBPβ. However, it was able to exhibit similar or even higher transcription levels by Tat compared to the parental LTR. C/EBPβ and Tat together further enhanced the transcription level of the parental LAI-LTR and DS3-9C LTR, with higher levels in the DS3-9C LTR. HIV molecular clone viruses carrying the DS3-9C variant LTR demonstrated a decreased replication capacity and delayed rate of replication. These results suggest that DS3 plays a role in virus transcriptional initiation and provides new insight into C/EBP regulation of HIV-1.

Sign in / Sign up

Export Citation Format

Share Document