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Helicobacter pylori FlgR Is an Enhancer-Independent Activator of  54-RNA
Polymerase Holoenzyme.Priyanka Brahmachary, 2004.Helicobacter pylori FlgR activates transcription with 54-RNA
polymerase holoenzyme (54-holoenzyme)
from at least five flagellar operons . Activators of
54-holoenzyme
generally bind enhancer sequences located >70 bp upstream of the
promoter and contact
54-holoenzyme
bound at the promoter through DNA looping to activate transcription.
H . pylori FlgR lacks the carboxy-terminal DNA-binding domain
present in most
54-dependent
activators . As little as 42 bp of DNA upstream of the flaB
promoter and 26 bp of DNA sequence downstream of the transcriptional
start site were sufficient for efficient FlgR-mediated expression
from a flaB'-'xylE reporter gene in H . pylori,
indicating that FlgR does not use an enhancer to activate
transcription . Other examples of
54-dependent
activators that lack a DNA-binding domain include Chlamydia
trachomatis CtcC and activators from the other Chlamydia
spp . whose genomes have been sequenced . FlgR from Helicobacter
hepaticus and Campylobacter jejuni, which are closely
related to H . pylori, appear to have carboxy-terminal
DNA-binding domains, suggesting that the loss of the DNA-binding
domain from H . pylori FlgR occurred after the divergence of
these bacterial species . Removal of the amino-terminal regulatory
domain of FlgR resulted in a constitutively active form of the
protein that activated transcription from
54-dependent
genes in Escherichia coli . The truncated FlgR protein also
activated transcription with E . coli
54-holoenzyme
in an in vitro transcription assay .
Unraveling the Mode of Action of the Antimalarial Choline Analog G25 in Plasmodium falciparum and Saccharomyces cerevisiae. Rodolphe Roggero, 2004.Pharmacological studies have indicated that the choline analog G25 is a potent inhibitor of Plasmodium falciparum growth in vitro and in vivo . Although choline transport has been suggested to be the target of G25, the exact mode of action of this compound is not known . Here we show that, similar to its effects on P . falciparum, G25 prevents choline entry into Saccharomyces cerevisiae cells and inhibits S . cerevisiae growth . However, we show that the uptake of this compound is not mediated by the choline carrier Hnm1 . An hnm1  yeast mutant, which lacks the only choline transporter gene HNM1, was not altered in the transport of a labeled analog of this compound . Eleven yeast mutants lacking genes involved in different steps of phospholipid biosynthesis were analyzed for their sensitivity to G25 . Four mutants affected in the de novo cytidyldiphosphate-choline-dependent phosphatidylcholine biosynthetic pathway and, surprisingly, a mutant strain lacking the phosphatidylserine decarboxylase-encoding gene PSD1 (but not PSD2) were found to be highly resistant to this compound . Based on these data for S . cerevisiae, labeling studies in P . falciparum were performed to examine the effect of G25 on the biosynthetic pathways of the major phospholipids phosphatidylcholine and phosphatidylethanolamine . Labeling studies in P . falciparum and in vitro studies with recombinant P . falciparum phosphatidylserine decarboxylase further supported the inhibition of both the de novo phosphatidylcholine metabolic pathway and the synthesis of phosphatidylethanolamine from phosphatidylserine . Together, our data indicate that G25 specifically targets the pathways for synthesis of the two major phospholipids, phosphatidylcholine and phosphatidylethanolamine, to exert its antimalarial activity .
Growth of Mycobacteria on Carbon Monoxide and Methanol. Sae W. Park, 2003.Several mycobacterial strains, such as Mycobacterium flavescens, Mycobacterium gastri, Mycobacterium neoaurum, Mycobacterium parafortuitum, Mycobacterium peregrinum, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium tuberculosis, and Mycobacterium vaccae, were found to grow on carbon monoxide (CO) as the sole source of carbon and energy . These bacteria, except for M . tuberculosis, also utilized methanol as the sole carbon and energy source . A CO dehydrogenase (CO-DH) assay, staining by activity of CO-DH, and Western blot analysis using an antibody raised against CO-DH of Mycobacterium sp . strain JC1 (formerly Acinetobacter sp . strain JC1 [J . W . Cho, H . S . Yim, and Y . M . Kim, Kor . J . Microbiol . 23:1-8, 1985]) revealed that CO-DH is present in extracts of the bacteria prepared from cells grown on CO . Ribulose bisphosphate carboxylase/oxygenase (RubisCO) activity was also detected in extracts prepared from all cells, except M . tuberculosis, grown on CO . The mycobacteria grown on methanol, except for M . gastri, which showed hexulose phosphate synthase activity, did not exhibit activities of classic methanol dehydrogenase, hydroxypyruvate reductase, or hexulose phosphate synthase but exhibited N,N-dimethyl-4-nitrosoaniline-dependent methanol dehydrogenase and RuBisCO activities . Cells grown on methanol were also found to have dihydroxyacetone synthase . Double immunodiffusion revealed that the antigenic sites of CO-DHs, RuBisCOs, and dihydroxyacetone synthases in all mycobacteria tested are identical with those of the Mycobacterium sp . strain JC1 enzymes . Detection of Staphylococcal Enterotoxin B via Biomolecular Interaction Analysis Mass Spectrometry. Dobrin Nedelkov, 2003.Detection of Staphylococcus enterotoxin B (SEB) by biomolecular interaction analysis mass spectrometry (BIA/MS) is presented in this work . The BIA/MS experiments were based on a surface plasmon resonance (SPR) MS immunoassay that detects affinity-captured SEB both via SPR and by means of exact and direct mass measurement by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry . Experiments were performed with standard samples and food samples to assess the BIA/MS limit of detection for SEB and to set the experimental parameters for proper quantitation . Single and double SPR referencing was performed to accurately estimate the amount of the bound toxin . Reproducible detection of 1 ng of SEB per ml, corresponding to affinity capture and MS analysis of  500 amol of SEB, was readily achieved from both the standard and mushroom samples . A certain amount of SEB degradation was indicated by the signals in the mass spectra . The combination of MS with SPR-based methods of detection creates a unique approach capable of quantifying and qualitatively analyzing protein toxins from pathogenic organisms .
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