scholarly journals plcR papR-Independent Expression of Anthrolysin O by Bacillus anthracis

2006 ◽  
Vol 188 (22) ◽  
pp. 7823-7829 ◽  
Author(s):  
Caná L. Ross ◽  
Theresa M. Koehler
Keyword(s):  
Bacillus Anthracis ◽  
Transcriptional Activation ◽  
Consensus Sequence ◽  
Gram Positive Bacteria ◽  
Significant Amino Acid Sequence ◽  
Steady State Levels ◽  
In Vivo Analysis ◽  
Translational Start ◽  
Anthrolysin O

ABSTRACT Cholesterol-dependent cytolysins (CDCs) are secreted, pore-forming toxins that are associated with pathogenesis in a variety of gram-positive bacteria. Bacillus anthracis produces anthrolysin O (ALO), a CDC that is largely responsible for the hemolytic activity of culture supernates when the bacterium is cultured in appropriate conditions. B. cereus and B. thuringiensis, species closely related to B. anthracis, produce CDCs with significant amino acid sequence homology to ALO. Transcription of the B. cereus and B. thuringiensis CDC genes is controlled by PlcR, a transcription regulator that requires a pentapeptide derived from the papR gene product for binding to a consensus sequence (PlcR box) and transcriptional activation of downstream genes. A PlcR box precedes the B. anthracis alo gene, and the B. anthracis genome contains three plcR-like genes, one of which harbors a nonsense mutation that is predicted to result in a truncated, nonfunctional protein. We detected mRNA of alo, papR, and the three plcR-like genes in spleens of B. anthracis-infected mice, indicating gene expression in vivo. Analysis of alo transcription in batch culture revealed a potential transcription start located between the PlcR box and the translational start. Nevertheless, steady-state levels of alo transcripts and ALO protein were unaffected by deletion of papR or disruption of the PlcR box. Our data indicate that despite the presence of the transcriptionally active plcR and papR genes in B. anthracis and a PlcR box in the promoter region of the alo gene, alo expression is independent of this control system.

scholarly journals Activation of the latent PlcR regulon in Bacillus anthracis

Microbiology ◽  
2010 ◽  
Vol 156 (10) ◽  
pp. 2982-2993 ◽  
Author(s):  
Inka Sastalla ◽  
Lauren M. Maltese ◽  
Olga M. Pomerantseva ◽  
Andrei P. Pomerantsev ◽  
Andrea Keane-Myers ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Consensus Sequence ◽  
Transcriptional Analysis ◽  
Virulence Plasmid ◽  
Putative Protease ◽  
2D Gel ◽  
Culture Supernatants ◽  
Anthrolysin O

Many genes in Bacillus cereus and Bacillus thuringiensis are under the control of the transcriptional regulator PlcR and its regulatory peptide, PapR. In Bacillus anthracis, the causative agent of anthrax, PlcR is inactivated by truncation, and consequently genes having PlcR binding sites are expressed at very low levels when compared with B. cereus. We found that activation of the PlcR regulon in B. anthracis by expression of a PlcR–PapR fusion protein does not alter sporulation in strains containing the virulence plasmid pXO1 and thereby the global regulator AtxA. Using comparative 2D gel electrophoresis, we showed that activation of the PlcR regulon in B. anthracis leads to upregulation of many proteins found in the secretome of B. cereus, including phospholipases and proteases, such as the putative protease BA1995. Transcriptional analysis demonstrated expression of BA1995 to be dependent on PlcR–PapR, even though the putative PlcR recognition site of the BA1995 gene does not exactly match the PlcR consensus sequence, explaining why this protein had escaped recognition as belonging to the PlcR regulon. Additionally, while transcription of major PlcR-dependent haemolysins, sphingomyelinase and anthrolysin O is enhanced in response to PlcR activation in B. anthracis, only anthrolysin O contributes significantly to lysis of human erythrocytes. In contrast, the toxicity of bacterial culture supernatants from a PlcR-positive strain towards murine macrophages occurred independently of anthrolysin O expression in vitro and in vivo.

scholarly journals Cyclic AMP-dependent protein kinase inhibits the activity of myogenic helix-loop-helix proteins.

1992 ◽  
Vol 12 (10) ◽  
pp. 4478-4485 ◽  
Author(s):  
L Li ◽  
R Heller-Harrison ◽  
M Czech ◽  
E N Olson
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Transcriptional Activation ◽  
Consensus Sequence ◽  
Signal Transduction Pathway ◽  
Specific Gene ◽  
Dependent Protein Kinase ◽  
Dependent Protein

Differentiation of skeletal muscle cells is inhibited by the cyclic AMP (cAMP) signal transduction pathway. Here we report that the catalytic subunit of cAMP-dependent protein kinase (PKA) can substitute for cAMP and suppress muscle-specific transcription by silencing the activity of the MyoD family of regulatory factors, which includes MyoD, myogenin, myf5, and MRF4. Repression by the PKA catalytic (C) subunit is directed at the consensus sequence CANNTG, the target for DNA binding and transcriptional activation by these myogenic regulators. Phosphopeptide mapping of myogenin in vitro and in vivo revealed two PKA phosphorylation sites, both within the basic region. However, repression of myogenin function by PKA does not require direct phosphorylation of these sites but instead involves an indirect mechanism with one or more intermediate steps. Regulation of the transcriptional activity of the MyoD family by modulation of the cAMP signaling pathway may account for the inhibitory effects of certain peptide growth factors on muscle-specific gene expression and may also determine the responsiveness of different cell types to myogenic conversion by these myogenic regulators.

N6-methyladenosine residues in an intron-specific region of prolactin pre-mRNA

1990 ◽  
Vol 10 (9) ◽  
pp. 4456-4465
Author(s):  
S M Carroll ◽  
P Narayan ◽  
F M Rottman
Keyword(s):  
Consensus Sequence ◽  
Specific Sequence ◽  
Free System ◽  
Consensus Sequences ◽  
Neplanocin A ◽  
A Cell ◽  
Steady State Levels ◽  
Precursor Rna

N6-methyladenosine (m6A) residues occur at internal positions in most cellular and viral RNAs; both heterogeneous nuclear RNA and mRNA are involved. This modification arises by enzymatic transfer of a methyl group from S-adenosylmethionine to the central adenosine residue in the canonical sequence G/AAC. Thus far, m6A has been mapped to specific locations in eucaryotic mRNA and viral genomic RNA. We have now examined an intron-specific sequence of a modified bovine prolactin precursor RNA for the presence of this methylated nucleotide by using both transfected-cell systems and a cell-free system capable of methylating mRNA transcripts in vitro. The results indicate the final intron-specific sequence (intron D) of a prolactin RNA molecule does indeed possess m6A residues. When mapped to specific T1 oligonucleotides, the predominant site of methylation was found to be within the consensus sequence AGm6ACU. The level of m6A at this site is nonstoichiometric; approximately 24% of the molecules are modified in vivo. Methylation was detected at markedly reduced levels at other consensus sites within the intron but not in T1 oligonucleotides which do not contain either AAC or GAC consensus sequences. In an attempt to correlate mRNA methylation with processing, stably transfected CHO cells expressing augmented levels of bovine prolactin were treated with neplanocin A, an inhibitor of methylation. Under these conditions, the relative steady-state levels of the intron-containing nuclear precursor increased four to six times that found in control cells.

Cyclic AMP-dependent protein kinase inhibits the activity of myogenic helix-loop-helix proteins

1992 ◽  
Vol 12 (10) ◽  
pp. 4478-4485
Author(s):  
L Li ◽  
R Heller-Harrison ◽  
M Czech ◽  
E N Olson
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Transcriptional Activation ◽  
Consensus Sequence ◽  
Signal Transduction Pathway ◽  
Specific Gene ◽  
Dependent Protein Kinase ◽  
Dependent Protein

Differentiation of skeletal muscle cells is inhibited by the cyclic AMP (cAMP) signal transduction pathway. Here we report that the catalytic subunit of cAMP-dependent protein kinase (PKA) can substitute for cAMP and suppress muscle-specific transcription by silencing the activity of the MyoD family of regulatory factors, which includes MyoD, myogenin, myf5, and MRF4. Repression by the PKA catalytic (C) subunit is directed at the consensus sequence CANNTG, the target for DNA binding and transcriptional activation by these myogenic regulators. Phosphopeptide mapping of myogenin in vitro and in vivo revealed two PKA phosphorylation sites, both within the basic region. However, repression of myogenin function by PKA does not require direct phosphorylation of these sites but instead involves an indirect mechanism with one or more intermediate steps. Regulation of the transcriptional activity of the MyoD family by modulation of the cAMP signaling pathway may account for the inhibitory effects of certain peptide growth factors on muscle-specific gene expression and may also determine the responsiveness of different cell types to myogenic conversion by these myogenic regulators.

scholarly journals Participation of the yeast activator Abf1 in meiosis-specific expression of the HOP1 gene.

1996 ◽  
Vol 16 (6) ◽  
pp. 2777-2786 ◽  
Author(s):  
V Gailus-Durner ◽  
J Xie ◽  
C Chintamaneni ◽  
A K Vershon
Keyword(s):  
Transcriptional Activation ◽  
Mutational Analysis ◽  
Consensus Sequence ◽  
Promoter Sequence ◽  
Regulatory Elements ◽  
Specific Gene ◽  
Specific Expression ◽  
Autonomously Replicating Sequence

The meiosis-specific gene HOP1, which encodes a component of the synaptonemal complex, is controlled through two regulatory elements, UASH and URS1H. Sites similar to URS1H have been identified in the promoter region of virtually every early meiosis-specific gene, as well as in many promoters of nonmeiotic genes, and it has been shown that the proteins that bind to this site function to regulate meiotic and nonmeiotic transcription. Sites similar to the UASH site have been found in a number of meiotic and nonmeiotic genes as well. Since it has been shown that UASH functions as an activator site in vegetative haploid cells, it seemed likely that the factors binding to this site regulate both meiotic and nonmeiotic transcription. We purified the factor binding to the UASH element of the HOP1 promoter. Sequence analysis identified the protein as Abf1 (autonomously replicating sequence-binding factor 1), a multifunctional protein involved in DNA replication, silencing, and transcriptional regulation. We show by mutational analysis of the UASH site, that positions outside of the proposed UASH consensus sequence (TNTGN[A/T]GT) are required for DNA binding in vitro and transcriptional activation in vivo. A new UASH consensus sequence derived from this mutational analysis closely matches a consensus Abf1 binding site. We also show that an Abf1 site from a nonmeiotic gene can replace the function of the UASH site in the HOP1 promoter. Taken together, these results show that Abf1 functions to regulate meiotic gene expression.

scholarly journals Activity of Dalbavancin against Bacillus anthracis In Vitro and in a Mouse Inhalation Anthrax Model

2010 ◽  
Vol 54 (3) ◽  
pp. 991-996 ◽  
Author(s):  
Henry S. Heine ◽  
Bret K. Purcell ◽  
Jennifer Bassett ◽  
Lynda Miller ◽  
Beth P. Goldstein
Keyword(s):  
Bacillus Anthracis ◽  
Half Life ◽  
Lethal Dose ◽  
Gram Positive Bacteria ◽  
Long Duration ◽  
Inhalation Anthrax ◽  
Duration Of Efficacy ◽  
Ames Strain

ABSTRACT Bacillus anthracis, the causative agent of anthrax, can produce fatal disease when it is inhaled or ingested by humans. Dalbavancin, a novel, semisynthetic lipoglycopeptide, has potent activity, greater than that of vancomycin, against Gram-positive bacteria and a half-life in humans that supports once-weekly dosing. Dalbavancin demonstrated potent in vitro activity against B. anthracis (MIC range, ≤0.03 to 0.5 mg/liter; MIC50 and MIC90, 0.06 and 0.25 mg/liter, respectively), which led us to test its efficacy in a murine inhalation anthrax model. The peak concentrations of dalbavancin in mouse plasma after the administration of single intraperitoneal doses of 5 and 20 mg/kg of body weight were 15 and 71 mg/kg, respectively. At 20 mg/kg, the dalbavancin activity was detectable for 6 days after administration (terminal half-life, 53 h), indicating that long intervals between doses were feasible. The mice were challenged with 50 to 100 times the median lethal dose of the Ames strain of B. anthracis, an inoculum that kills untreated animals within 4 days. The efficacy of dalbavancin was 80 to 100%, as determined by the rate of survival at 42 days, when treatment was initiated 24 h postchallenge with regimens of 15 to 120 mg/kg every 36 h (q36h) or 30 to 240 mg/kg every 72 h (q72h). A regimen of ciprofloxacin known to protect 100% of animals was tested in parallel. Delayed dalbavancin treatment (beginning 36 or 48 h postchallenge) with 60 mg/kg q36h or 120 mg/kg q72h still provided 70 to 100% survival. The low MICs and long duration of efficacy in vivo suggest that dalbavancin may have potential as an alternative treatment or for the prophylaxis of B. anthracis infections.

scholarly journals N6-methyladenosine residues in an intron-specific region of prolactin pre-mRNA.

1990 ◽  
Vol 10 (9) ◽  
pp. 4456-4465 ◽  
Author(s):  
S M Carroll ◽  
P Narayan ◽  
F M Rottman
Keyword(s):  
Consensus Sequence ◽  
Specific Sequence ◽  
Free System ◽  
Consensus Sequences ◽  
Neplanocin A ◽  
A Cell ◽  
Steady State Levels ◽  
Precursor Rna

N6-methyladenosine (m6A) residues occur at internal positions in most cellular and viral RNAs; both heterogeneous nuclear RNA and mRNA are involved. This modification arises by enzymatic transfer of a methyl group from S-adenosylmethionine to the central adenosine residue in the canonical sequence G/AAC. Thus far, m6A has been mapped to specific locations in eucaryotic mRNA and viral genomic RNA. We have now examined an intron-specific sequence of a modified bovine prolactin precursor RNA for the presence of this methylated nucleotide by using both transfected-cell systems and a cell-free system capable of methylating mRNA transcripts in vitro. The results indicate the final intron-specific sequence (intron D) of a prolactin RNA molecule does indeed possess m6A residues. When mapped to specific T1 oligonucleotides, the predominant site of methylation was found to be within the consensus sequence AGm6ACU. The level of m6A at this site is nonstoichiometric; approximately 24% of the molecules are modified in vivo. Methylation was detected at markedly reduced levels at other consensus sites within the intron but not in T1 oligonucleotides which do not contain either AAC or GAC consensus sequences. In an attempt to correlate mRNA methylation with processing, stably transfected CHO cells expressing augmented levels of bovine prolactin were treated with neplanocin A, an inhibitor of methylation. Under these conditions, the relative steady-state levels of the intron-containing nuclear precursor increased four to six times that found in control cells.

scholarly journals Transcriptional Activation of the Aldehyde Reductase YqhD by YqhC and Its Implication in Glyoxal Metabolism of Escherichia coli K-12

2010 ◽  
Vol 192 (16) ◽  
pp. 4205-4214 ◽  
Author(s):  
Changhan Lee ◽  
Insook Kim ◽  
Junghoon Lee ◽  
Kang-Lok Lee ◽  
Bumchan Min ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Transcriptional Activation ◽  
Consensus Sequence ◽  
Aldehyde Reductase ◽  
Reverse Transcription Pcr ◽  
Reduction Activity ◽  
Enzymatic Reduction ◽  
K 12 ◽  
Mutant Forms

ABSTRACT The reactive α-oxoaldehydes such as glyoxal (GO) and methylglyoxal (MG) are generated in vivo from sugars through oxidative stress. GO and MG are believed to be removed from cells by glutathione-dependent glyoxalases and other aldehyde reductases. We isolated a number of GO-resistant (GOr) mutants from Escherichia coli strain MG1655 on LB plates containing 10 mM GO. By tagging the mutations with the transposon TnphoA-132 and determining their cotransductional linkages, we were able to identify a locus to which most of the GOr mutations were mapped. DNA sequencing of the locus revealed that it contains the yqhC gene, which is predicted to encode an AraC-type transcriptional regulator of unknown function. The GOr mutations we identified result in missense changes in yqhC and were concentrated in the predicted regulatory domain of the protein, thereby constitutively activating the product of the adjacent gene yqhD. The transcriptional activation of yqhD by wild-type YqhC and its mutant forms was established by an assay with a β-galactosidase reporter fusion, as well as with real-time quantitative reverse transcription-PCR. We demonstrated that YqhC binds to the promoter region of yqhD and that this binding is abolished by a mutation in the potential target site, which is similar to the consensus sequence of its homolog SoxS. YqhD facilitates the removal of GO through its NADPH-dependent enzymatic reduction activity by converting it to ethadiol via glycolaldehyde, as detected by nuclear magnetic resonance, as well as by spectroscopic measurements. Therefore, we propose that YqhC is a transcriptional activator of YqhD, which acts as an aldehyde reductase with specificity for certain aldehydes, including GO.

scholarly journals Transcriptional Activation of the Bacillus subtilis ackA Gene Requires Sequences Upstream of the Promoter

1998 ◽  
Vol 180 (22) ◽  
pp. 5961-5967 ◽  
Author(s):  
Andrew J. Turinsky ◽  
Frank J. Grundy ◽  
Jeong-Ho Kim ◽  
Glenn H. Chambliss ◽  
Tina M. Henkin
Keyword(s):  
Bacillus Subtilis ◽  
Carbon Source ◽  
Transcriptional Activation ◽  
Consensus Sequence ◽  
Protein A ◽  
Acetate Kinase ◽  
Gene Encoding ◽  
Gram Positive Bacteria ◽  
Catabolite Control Protein A

ABSTRACT Transcriptional activation of the Bacillus subtilis ackA gene, encoding acetate kinase, was previously shown to require catabolite control protein A (CcpA) and sequences upstream of the ackA promoter. CcpA, which is responsible for catabolite repression of a number of secondary carbon source utilization genes in B. subtilis and other gram-positive bacteria, recognizes a cis-acting consensus sequence, designated cre (catabolite response element), generally located within or downstream of the promoter of the repressed gene. Two sites resembling this sequence are centered at positions −116.5 and −56.5 of the ackA promoter and have been termedcre1 and cre2, respectively. Synthesis of acetate kinase, which is involved in the conversion of acetyl coenzyme A to acetate, is induced when cells are grown in the presence of an easily metabolized carbon source such as glucose. In this study,cre2, the site closer to the promoter, and the region upstream of cre2 were shown to be indispensable for CcpA-dependent transcriptional activation of ackA, whereascre1 was not required. In addition, insertion of 5 bp between cre2 and the promoter disrupted activation, while 10 bp was tolerated, suggesting face-of-the-helix dependence of the position of cre2 and/or upstream sequences. DNase footprinting experiments demonstrated binding of CcpA in vitro tocre2 but not cre1, consistent with the genetic data. Activation of ackA transcription was blocked in aptsH1/crh double mutant, suggesting involvement of this pathway in CcpA-mediated transcriptional activation.

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