scholarly journals Dam- and OxyR-Dependent Phase Variation of agn43: Essential Elements and Evidence for a New Role of DNA Methylation

2002 ◽  
Vol 184 (12) ◽  
pp. 3338-3347 ◽  
Author(s):  
Anu Wallecha ◽  
Vincent Munster ◽  
Jason Correnti ◽  
Teresa Chan ◽  
Marjan van der Woude
Keyword(s):  
Dna Methylation ◽  
Regulatory Region ◽  
Phase Variation ◽  
Essential Elements ◽  
Reduced Form ◽  
Transcription In Vitro ◽  
Target Sequences

ABSTRACT Phase variation of the outer membrane protein Ag43 in E. coli requires deoxyadenosine methylase (Dam) and OxyR. Previously, it was shown that OxyR is required for repression of the Ag43-encoding gene, agn43, and that Dam-dependent methylation of three GATC target sequences in the regulatory region abrogates OxyR binding. Here we report further characterization of agn43 transcription and its regulation. Transcription was initiated from a σ70-dependent promoter at the G residue of the upstream GATC sequence. Template DNA and RNA polymerase were sufficient to obtain transcription in vitro, but DNA methylation enhanced the level of transcription. Analyses of transcription in vivo of agn′-lacZ with mutated Dam target sequences support this conclusion. Since methylation also abrogates OxyR binding, this indicates that methylation plays a dual role in facilitating agn43 transcription. In vitro transcription from an unmethylated template was repressed by OxyR(C199S), which resembles the reduced form of OxyR. Consistent with this and the role of Dam in OxyR binding, OxyR(C199S) protected from DNase I digestion the agn43 regulatory region from −16 to +42, which includes the three GATC sequences. Deletion analyses of the regulatory region showed that a 101-nucleotide region of the agn43 regulatory region containing the promoter and this OxyR binding region was sufficient for Dam- and OxyR-dependent phase variation

scholarly journals Phase Variation of Ag43 Is Independent of the Oxidation State of OxyR

2003 ◽  
Vol 185 (7) ◽  
pp. 2203-2209 ◽  
Author(s):  
Anu Wallecha ◽  
Jason Correnti ◽  
Vincent Munster ◽  
Marjan van der Woude
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Oxidation State ◽  
Regulatory Region ◽  
Phase Variation ◽  
In Vitro Transcription ◽  
Reduced Form ◽  
Target Sequences

ABSTRACT OxyR is a DNA binding protein that differentially regulates a cell's response to hydrogen peroxide-mediated oxidative stress. We previously reported that the reduced form of OxyR is sufficient for repression of transcription of agn43 from unmethylated template DNA, which is essential for deoxyadenosine methylase (Dam)- and OxyR-dependent phase variation of agn43. Here we provide evidence that the oxidized form of OxyR [OxyR(ox)] also represses agn43 transcription. In vivo, we found that exogenous addition of hydrogen peroxide, sufficient to oxidize OxyR, did not affect the expression of agn43. OxyR(ox) repressed in vitro transcription but only from an unmethylated agn43 template. The −10 sequence of the promoter and three Dam target sequences were protected in an in vitro DNase I footprint assay by OxyR(ox). Furthermore, OxyR(ox) bound to the agn43 regulatory region DNA with an affinity similar to that for the regulatory regions of katG and oxyS, which are activated by OxyR(ox), indicating that binding at agn43 can occur at biologically relevant concentrations. OxyR-dependent regulation of Ag43 expression is therefore unusual in firstly that OxyR binding at agn43 is dependent on the methylation state of Dam target sequences in its binding site and secondly that OxyR-dependent repression appears to be independent of hydrogen-peroxide mediated oxidative stress and the oxidation state of OxyR.

scholarly journals Transcription factor MBF-I interacts with metal regulatory elements of higher eucaryotic metallothionein genes.

1989 ◽  
Vol 9 (12) ◽  
pp. 5315-5323 ◽  
Author(s):  
J Imbert ◽  
M Zafarullah ◽  
V C Culotta ◽  
L Gedamu ◽  
D Hamer
Keyword(s):  
Gene Transcription ◽  
Regulatory Elements ◽  
Gene Promoters ◽  
Direct Role ◽  
Transcription In Vitro ◽  
Mouse Cells ◽  
Affinity Reagent

Metallothionein (MT) gene promoters in higher eucaryotes contain multiple metal regulatory elements (MREs) that are responsible for the metal induction of MT gene transcription. We identified and purified to near homogeneity a 74-kilodalton mouse nuclear protein that specifically binds to certain MRE sequences. This protein, MBF-I, was purified employing as an affinity reagent a trout MRE that is shown to be functional in mouse cells but which lacks the G+C-rich and SP1-like sequences found in many mammalian MT gene promoters. Using point-mutated MREs, we showed that there is a strong correlation between DNA binding in vitro and MT gene regulation in vivo, suggesting a direct role of MBF-I in MT gene transcription. We also showed that MBF-I can induce MT gene transcription in vitro in a mouse extract and that this stimulation requires zinc.

scholarly journals Role of Endoproteolytic Dibasic Proprotein Processing in Maturation of Secretory Proteins inTrichoderma reesei

1998 ◽  
Vol 64 (9) ◽  
pp. 3202-3208 ◽  
Author(s):  
Sabine P. Goller ◽  
Doris Schoisswohl ◽  
Michel Baron ◽  
Martine Parriche ◽  
Christian P. Kubicek
Keyword(s):  
Secretory Proteins ◽  
Heterologous Proteins ◽  
Homologous Proteins ◽  
Cell Extracts ◽  
Endopeptidase Activity ◽  
Proprotein Processing ◽  
Target Sequences

ABSTRACT Cell extracts of Trichoderma reesei exhibited dibasic endopeptidase activity toward the carboxylic side of KR, RR, and PR sequences. This activity was stimulated by the presence of Ca2+ ions and localized in vesicles of low bouyant density; it therefore exhibited some similarity to yeast Kex2. Analytical chromatofocusing revealed a single peak of activity. The dibasic endopeptidase activity was strongly and irreversibly inhibited in vitro as well as in vivo by 1 mM p-amidinophenylmethylsulfonyl fluoride (pAPMSF) but not by PMSF at concentrations up to 5 mM. We therefore used pAPMSF to study the role of the dibasic endopeptidase in the secretion of protein by T. reesei. Secretion of xylanase I (proprotein processing sequence -R-R-↓-R-↓-A-) and xylanase II (-K-R-↓-Q-) was strongly inhibited by 1 mM pAPMSF, and a larger, unprocessed enzyme form was detected intracellularly under these conditions. Secretion of cellobiohydrolase II (CBH II; -E-R-↓-Q-) was only slightly inhibited by pAPMSF, and no accumulation of unprocessed precursors was detected. In contrast, secretion of CBH I (-R-A-↓-Q-) was stimulated by pAPMSF addition, and a simultaneous decrease in the concentration of intracellular CBH I was detected. Similar experiments were also carried out with a single heterologous protein, ShBLE, the phleomycin-binding protein fromStreptoalloteichus hindustanus, fused to a series of model proprotein-processing sequences downstream of the expression signals of the Aspergillus nidulans gpdA promoter. Consistent with the results obtained with homologous proteins, pAPMSF inhibited the secretion of ShBLE with fusions containing dibasic (RK and KR) target sequences, but it even stimulated secretion in fusions to LR, NHA, and EHA target sequences. Addition of 5 mM PMSF, a nonspecific inhibitor of serine protease, nonspecifically inhibited the secretion of heterologous proteins from fusions bearing the NHA and LR targets. These data point to the existence of different endoproteolytic proprotein processing enzymes in T. reesei and demonstrate that dibasic processing is obligatory for the secretion of the proproteins containing this target.

scholarly journals Formation of DNA Methylation Patterns: Nonmethylated GATC Sequences in gut and papOperons

1998 ◽  
Vol 180 (22) ◽  
pp. 5913-5920 ◽  
Author(s):  
Marjan van der Woude ◽  
W. Bradley Hale ◽  
David A. Low
Keyword(s):  
Dna Methylation ◽  
Regulatory Protein ◽  
Regulatory Region ◽  
Published Data ◽  
Catabolite Gene Activator Protein ◽  
A Site ◽  
Methylation Patterns ◽  
Gatc Site

ABSTRACT Most of the adenine residues in GATC sequences in theEscherichia coli chromosome are methylated by the enzyme deoxyadenosine methyltransferase (Dam). However, at least 20 GATC sequences remain nonmethylated throughout the cell cycle. Here we examined how the DNA methylation patterns of GATC sequences within the regulatory regions of the pyelonephritis-associated pilus (pap) operon and the glucitol utilization (gut) operon were formed. The results obtained with an in vitro methylation protection assay showed that the addition of the leucine-responsive regulatory protein (Lrp) to pap DNA was sufficient to protect the two GATC sequences in the pap regulatory region, GATC-I and GATC-II, from methylation by Dam. This finding was consistent with previously published data showing that Lrp was essential for methylation protection of these DNA sites in vivo. Methylation protection also occurred at a GATC site (GATC-44.5) centered 44.5 bp upstream of the transcription start site of thegutABD operon. Two proteins, GutR and the catabolite gene activator protein (CAP), bound to DNA sites overlapping the GATC-44.5-containing region of the gutABD operon. GutR, an operon-specific repressor, was essential for methylation protection in vivo, and binding of GutR protected GATC-44.5 from methylation in vitro. In contrast, binding of CAP at a site overlapping GATC-44.5 did not protect this site from methylation. Mutational analyses indicated that gutABD gene regulation was not controlled by methylation of GATC-44.5, in contrast to regulation of Pap pilus expression, which is directly controlled by methylation of thepap GATC-I and GATC-II sites.

scholarly journals Use of a Two-Color Genetic Screen To Identify a Domain of the Global Regulator Lrp That Is Specifically Required forpap Phase Variation

1998 ◽  
Vol 180 (5) ◽  
pp. 1224-1231 ◽  
Author(s):  
Linda Kaltenbach ◽  
Bruce Braaten ◽  
Julie Tucker ◽  
Margareta Krabbe ◽  
David Low
Keyword(s):  
Amino Acid ◽  
Binding Sites ◽  
Phase Variation ◽  
Genetic Screen ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Global Regulator ◽  
Transcription In Vitro

ABSTRACT The global regulator Lrp plays a central role as both a repressor and an activator in Pap phase variation. Unlike most other members of the Lrp regulon such as ilvIH, activation ofpapBA transcription requires the coregulator PapI and is methylation dependent. We developed a two-color genetic screen to identify Lrp mutations that inhibit Pap phase variation but still activate ilvIH transcription, reasoning that such mutations might identify PapI binding or methylation-responsive domains. Amino acid substitutions in Lrp at position 126, 133, or 134 greatly reduced the rate of Pap switching from phase off to phase on but had much smaller effects on ilvIH transcription. In vitro analyses indicated that the T134A and E133G Lrp variants maintained affinities for pap and ilvIH DNAs similar to those of wild-type Lrp. In addition, both mutant Lrp’s were as responsive to PapI as wild-type Lrp, evidenced by an increase in affinity forpap Lrp binding sites 4, 5, and 6. Thus, in vitro analyses did not reveal the step(s) in Pap phase variation where these Lrp mutants were inhibited. In vivo analyses showed that both the T134A and E133G Lrp mutants activated transcription of a phase-on-lockedpap derivative containing a mutation in Lrp binding site 3. Further studies indicated that the T134A Lrp mutant was blocked in a step in Pap phase variation that does not involve PapI. Our data suggest that these mutant Lrp’s are defective in a previously unidentified interaction required for the switch from the phase-off to the phase-on pap transcription state.

Transcription factor MBF-I interacts with metal regulatory elements of higher eucaryotic metallothionein genes

1989 ◽  
Vol 9 (12) ◽  
pp. 5315-5323
Author(s):  
J Imbert ◽  
M Zafarullah ◽  
V C Culotta ◽  
L Gedamu ◽  
D Hamer
Keyword(s):  
Gene Transcription ◽  
Regulatory Elements ◽  
Gene Promoters ◽  
Direct Role ◽  
Transcription In Vitro ◽  
Mouse Cells ◽  
Affinity Reagent

Metallothionein (MT) gene promoters in higher eucaryotes contain multiple metal regulatory elements (MREs) that are responsible for the metal induction of MT gene transcription. We identified and purified to near homogeneity a 74-kilodalton mouse nuclear protein that specifically binds to certain MRE sequences. This protein, MBF-I, was purified employing as an affinity reagent a trout MRE that is shown to be functional in mouse cells but which lacks the G+C-rich and SP1-like sequences found in many mammalian MT gene promoters. Using point-mutated MREs, we showed that there is a strong correlation between DNA binding in vitro and MT gene regulation in vivo, suggesting a direct role of MBF-I in MT gene transcription. We also showed that MBF-I can induce MT gene transcription in vitro in a mouse extract and that this stimulation requires zinc.

scholarly journals Autonomous Role of 3′-Terminal CCCA in Directing Transcription of RNAs by Qβ Replicase

2001 ◽  
Vol 75 (23) ◽  
pp. 11373-11383 ◽  
Author(s):  
David M. Tretheway ◽  
Shigeo Yoshinari ◽  
Theo W. Dreher
Keyword(s):  
Escherichia Coli ◽  
Secondary Structure ◽  
Similar Sequence ◽  
Transcriptional Initiation ◽  
Template Sequence ◽  
Transcription In Vitro ◽  
Internal Initiation

ABSTRACT We have studied transcription in vitro by Qβ replicase to deduce the minimal features needed for efficient end-to-end copying of an RNA template. Our studies have used templates ca. 30 nucleotides long that are expected to be free of secondary structure, permitting unambiguous analysis of the role of template sequence in directing transcription. A 3′-terminal CCCA (3′-CCCA) directs transcriptional initiation to opposite the underlined C; the amount of transcription is comparable between RNAs possessing upstream (CCA) n tracts, A-rich sequences, or a highly folded domain and is also comparable in single-round transcription assays to transcription of two amplifiable RNAs. Predominant initiation occurs within the 3′-CCCA initiation box when a wide variety of sequences is present immediately upstream, but CCA or a closely similar sequence in that position results in significant internal initiation. Removal of the 3′-A from the 3′-CCCA results in 5- to 10-fold-lower transcription, emphasizing the importance of the nontemplated addition of 3′-A by Qβ replicase during termination. In considering whether 3′-CCCA could provide sufficient specificity for viral transcription, and consequently amplification, in vivo, we note that tRNAHis is the only stable Escherichia coliRNA with 3′-CCCA. In vitro-generated transcripts corresponding to tRNAHis served as poor templates for Qβ replicase; this was shown to be due to the inaccessibility of the partially base-paired CCCA. These studies demonstrate that 3′-CCCA plays a major role in the control of transcription by Qβ replicase and that the abundant RNAs present in the host cell should not be efficient templates.

scholarly journals Transcriptional Organization and In Vivo Role of theEscherichia coli rsd Gene, Encoding the Regulator of RNA Polymerase Sigma D

1999 ◽  
Vol 181 (12) ◽  
pp. 3768-3776 ◽  
Author(s):  
Miki Jishage ◽  
Akira Ishihama
Keyword(s):  
Rna Polymerase ◽  
Stationary Phase ◽  
Null Mutant ◽  
Gene Encoding ◽  
Transcription In Vitro ◽  
Extension Analysis ◽  
Insight Into

ABSTRACT The regulator of sigma D (Rsd) was identified as an RNA polymerase ς70-associated protein in stationary-phaseEscherichia coli with the inhibitory activity of ς70-dependent transcription in vitro (M. Jishage and A. Ishihama, Proc. Natl. Acad. Sci. USA 95:4953–4958, 1998). Primer extension analysis of rsd mRNA indicated the presence of two promoters, ςS-dependent P1 and ς70-dependent P2 with the gearbox sequence. To get insight into the in vivo role of Rsd, the expression of a reporter gene fused to either the ς70- or ςS-dependent promoter was analyzed in the absence of Rsd or the presence of overexpressed Rsd. In the rsd null mutant, the ς70- and ςS-dependent gene expression was increased or decreased, respectively. On the other hand, the ς70- or ςS-dependent transcription was reduced or enhanced, respectively, after overexpression of Rsd. The repression of the ςS-dependent transcription in the rsd mutant is overcome by increased production of the ςS subunit. Together these observations support the prediction that Rsd is involved in replacement of the RNA polymerase ς subunit from ς70 to ςS during the transition from exponential growth to the stationary phase.

scholarly journals PhotorhabdusDam methyltransferase overexpression impairs virulence of the nemato-bacterial complex in insects

2019 ◽  
Author(s):  
Amaury Payelleville ◽  
Dana Blackburn ◽  
Anne Lanois ◽  
Sylvie Pagès ◽  
Marine Cambon ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Infective Juvenile ◽  
Photorhabdus Luminescens ◽  
Control Strain ◽  
Bacterial Dna ◽  
Bacterial Complex ◽  
Overexpressing Strain

AbstractPhotorhabdus luminescensis an entomopathogenic bacterium found in symbiosis with the nematodeHeterorhabditis. Dam DNA methylation is involved in the pathogenicity of many bacteria, includingP. luminescens,whereas studies about the role of bacterial DNA methylation during symbiosis are scarce. The aim of this study was to determine the role of Dam DNA methylation inP. luminescenssymbiosis withH. bacteriophora. We constructed a strain overexpressingdamby inserting an additional copy of thedamgene under the control of a constitutive promoter in the chromosome ofP. luminescensand then achieved association between this recombinant strain and nematodes. Thedamoverexpressing strain was able to feed the nematodein vitroandin vivosimilarly as a control strain, and to re-associate with Infective Juvenile (IJ) stages in the insect. No difference in the amount of emerging IJs from the cadaver was observed between the two strains. Compared to the nematode in symbiosis with the control strain, a significant increase in LT50was observed during insect infestation with the nematode associated with thedamoverexpressing strain. These results suggest that theP. luminescensDam plays a role in the pathogenicity of the nemato-bacterial complex.

Enhancer DNA methylation: implications for gene regulation

2019 ◽  
Vol 63 (6) ◽  
pp. 707-715 ◽  
Author(s):  
Allegra Angeloni ◽  
Ozren Bogdanovic
Keyword(s):  
Dna Methylation ◽  
Model Systems ◽  
Enhancer Activity ◽  
Cpg Dinucleotides ◽  
Germline Genes ◽  
Dna Elements

Abstract DNA methylation involves the addition of a methyl group to the fifth carbon of the pyrimidine cytosine ring (5-methylcytosine, 5mC). 5mC is widespread in vertebrate genomes where it is predominantly found within CpG dinucleotides. In mammals, 5mC participates in long-term silencing processes such as X-chromosome inactivation, genomic imprinting, somatic silencing of germline genes, and silencing of repetitive DNA elements. The evidence for 5mC as a dynamic gene-regulatory mechanism is mostly limited to specific examples, and is far from being completely understood. Recent work from diverse model systems suggests that 5mC might not always act as a dominant repressive mechanism and that hypermethylated promoters and enhancers can be permissive to transcription in vivo and in vitro. In this review, we discuss the links between 5mC and enhancer activity, and evaluate the role of this biochemical mechanism in various biological contexts.

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