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Regulation of the Tryptophan Biosynthetic Genes in Bacillus halodurans: Common Elements but Different Strategies than Those Used by Bacillus subtilis. Reka Szigeti, 2004.In Bacillus subtilis, an RNA binding protein called TRAP regulates both transcription and translation of the tryptophan biosynthetic genes . Bacillus halodurans is an alkaliphilic Bacillus species that grows at high pHs . Previous studies of this bacterium have focused on mechanisms of adaptation for growth in alkaline environments . We have characterized the regulation of the tryptophan biosynthetic genes in B . halodurans and compared it to that in B . subtilis . B . halodurans encodes a TRAP protein with 71% sequence identity to the B . subtilis protein . Expression of anthranilate synthetase, the first enzyme in the pathway to tryptophan, is regulated significantly less in B . halodurans than in B . subtilis . Examination of the control of the B . halodurans trpEDCFBA operon both in vivo and in vitro shows that only transcription is regulated, whereas in B . subtilis both transcription of the operon and translation of trpE are controlled . The attenuation mechanism that controls transcription in B . halodurans is similar to that in B . subtilis, but there are some differences in the predicted RNA secondary structures in the B . halodurans trp leader region, including the presence of a potential anti-antiterminator structure . Translation of trpG, which is within the folate operon in both bacilli, is regulated similarly in the two species . Molecular Mechanisms of Anti-Inflammatory Action of Erythromycin in Human Bronchial Epithelial Cells: Possible Role in the Signaling Pathway That Regulates Nuclear Factor-  B Activation.Masashi Desaki, 2004.Long-term macrolide therapy has been proven to improve survival in patients with diffuse panbronchiolitis . Although its mechanisms remain unknown, previous studies have suggested the effects of macrolide might be anti-inflammatory rather than antibacterial . To elucidate the molecular mechanisms of its action, we studied here the effects of erythromycin (EM) and its new derivative, EM703, which shows no antibacterial action, on the activation of the transcription factor nuclear factor- B (NF-B) in human bronchial epithelial cells . Western blotting analysis showed that EM did not inhibit the degradation of IB , suggesting the molecular target for EM was not the dissociation of NF-B from IB . An electrophoretic mobility shift assay showed that EM did not interrupt the NF-B DNA-binding activity in the nucleus under the conditions tested . Moreover, not only EM but also EM703 suppressed the activation of NF-B and the production of interleukin-8, demonstrating that the anti-inflammatory action of the macrolide is independent of its antibacterial activity . Taken together, these data suggest EM has an anti-inflammatory action, presumably via an interaction with the NF-B signaling pathway in the downstream of the dissociation from IB, resulting in the inhibition of NF-B .
Detection of Low Levels of Listeria monocytogenes Cells by Using a Fiber-Optic Immunosensor. Tao Geng, 2004.Biosensor technology has a great potential to meet the need for sensitive and nearly real-time microbial detection from foods . An antibody-based fiber-optic biosensor to detect low levels of Listeria monocytogenes cells following an enrichment step was developed . The principle of the sensor is a sandwich immunoassay where a rabbit polyclonal antibody was first immobilized on polystyrene fiber waveguides through a biotin-streptavidin reaction to capture Listeria cells on the fiber . Capture of cells on the fibers was confirmed by scanning electron microscopy . A cyanine 5-labeled murine monoclonal antibody, C11E9, was used to generate a specific fluorescent signal, which was acquired by launching a 635-nm laser light from an Analyte 2000 and collected by a photodetector at 670 to 710 nm . This immunosensor was specific for L . monocytogenes and showed a significantly higher signal strength than for other Listeria species or other microorganisms, including Escherichia coli, Enterococcus faecalis, Salmonella enterica, Lactobacillus plantarum, Carnobacterium gallinarum, Hafnia alvei, Corynebacterium glutamicum, Enterobacter aerogenes, Pseudomonas aeruginosa, and Serratia marcescens, in pure or in mixed-culture setup . Fiber-optic results could be obtained within 2.5 h of sampling . The sensitivity threshold was about 4.3 x 103 CFU/ml for a pure culture of L . monocytogenes grown at 37°C . When L . monocytogenes was mixed with lactic acid bacteria or grown at 10°C with 3.5% NaCl, the detection threshold was 4.1 x 104 or 2.8 x 107 CFU/ml, respectively . In less than 24 h, this method could detect L . monocytogenes in hot dog or bologna naturally contaminated or artificially inoculated with 10 to 1,000 CFU/g after enrichment in buffered Listeria enrichment broth . Detection of Other Microbial Species by Salmonella: Expression of the SdiA Regulon. Jenée N. Smith, 2003.Salmonella, Escherichia, and Klebsiella do not encode any recognized type of N-acylhomoserine lactone (AHL) synthase, and consistent with this, they do not synthesize AHLs under any conditions tested . However, they do encode an AHL receptor of the LuxR family, named SdiA . MudJ fusions in four loci are known to respond to plasmid-encoded sdiA in Salmonella, but only the rck locus has been described . Here we report the location and sequence analysis of the remaining three loci . The srg-6::MudJ is within gtgA of the gifsy-2 prophage, and the srg-7::MudJ is within PSLT61 of the virulence plasmid . Both fusions are in the antisense orientation . The third fusion, srgE5::MudJ, is within a horizontally acquired gene of unknown function at 33.6 centisomes that we have named srgE . Previously, sdiA expressed from its natural position in the chromosome was demonstrated to activate a plasmid-based transcriptional fusion to the rck promoter in response to AHL production by other bacterial species . However, the MudJ fusions did not respond to chromosomal sdiA . Here we report that MudJ fusions to three of the four loci (not srg-6) are activated by AHL in an sdiA-dependent manner during growth in motility agar (0.25% agar) but not during growth in top agar (0.7% agar) or on agar plates (1.2% agar) . In motility agar, the srgE promoter responds to sdiA at 30°C and higher while the rck and srg-7 promoters respond only at 37 or 42°C . Substantial AHL-independent SdiA activity was observed at 30°C but not at 37°C . Bacterial Community Dynamics during Production of Registered Designation of Origin Salers Cheese as Evaluated by 16S rRNA Gene Single-Strand Conformation Polymorphism Analysis. Frédérique Duthoit, 2003.Microbial dynamics during processing and ripening of traditional cheeses such as registered designation of origin Salers cheese, an artisanal cheese produced in France, play an important role in the elaboration of sensory qualities . The aim of the present study was to obtain a picture of the dynamics of the microbial ecosystem of RDO Salers cheese by using culture-independent methods . This included DNA extraction, PCR, and single-strand conformation polymorphism (SSCP) analysis . Bacterial and high-GC% gram-positive bacterial primers were used to amplify V2 or V3 regions of the 16S rRNA gene . SSCP patterns revealed changes during the manufacturing of the cheese . Patterns of the ecosystems of cheeses that were provided by three farmers were also quite different . Cloning and sequencing of the 16S rRNA gene revealed sequences related to lactic acid bacteria (Lactococcus lactis, Streptococcus thermophilus, Enterococcus faecium, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Lactobacillus plantarum, and Lactobacillus pentosus), which were predominant during manufacturing and ripening . Bacteria belonging to the high-GC% gram-positive group (essentially corynebacteria) were found by using specific primers . The present molecular approach can effectively describe the ecosystem of artisanal dairy products .
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