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Identification of AlgR-Regulated Genes in Pseudomonas aeruginosa by Use of Microarray Analysis. Stephen E. Lizewski, 2004.The Pseudomonas aeruginosa transcriptional regulator AlgR controls a variety of different processes, including alginate production, type IV pilus function, and virulence, indicating that AlgR plays a pivotal role in the regulation of gene expression . In order to characterize the AlgR regulon, Pseudomonas Affymetrix GeneChips were used to generate the transcriptional profiles of (i) P . aeruginosa PAO1 versus its algR mutant in mid-logarithmic phase, (ii) P . aeruginosa PAO1 versus its algR mutant in stationary growth phase, and (iii) PAO1 versus PAO1 harboring an algR overexpression plasmid . Expression analysis revealed that, during mid-logarithmic growth, AlgR activated the expression of 58 genes while it repressed the expression of 37 others, while during stationary phase, it activated expression of 45 genes and repression of 14 genes . Confirmatory experiments were performed on two genes found to be AlgR repressed (hcnA and PA1557) and one AlgR-activated operon (fimU-pilVWXY1Y2) . An S1 nuclease protection assay demonstrated that AlgR repressed both known hcnA promoters in PAO1 . Additionally, direct measurement of hydrogen cyanide (HCN) production showed that P . aeruginosa PAO1 produced threefold-less HCN than did its algR deletion strain . AlgR also repressed transcription of two promoters of the uncharacterized open reading frame PA1557 . Further, the twitching motility defect of an algR mutant was complemented by the fimTU-pilVWXY1Y2E operon, thus identifying the AlgR-controlled genes responsible for this defect in an algR mutant . This study identified four new roles for AlgR: (i) AlgR can repress gene transcription, (ii) AlgR activates the fimTU-pilVWXY1Y2E operon, (iii) AlgR regulates HCN production, and (iv) AlgR controls transcription of the putative cbb3-type cytochrome PA1557 . The Glycosyltransferase Gene Encoding the Enzyme Catalyzing the First Step of Mycothiol Biosynthesis (mshA). Gerald L. Newton, 2003.Mycothiol is the major thiol present in most actinomycetes and is produced from the pseudodisaccharide 1D-myo-inosityl 2-acetamido-2-deoxy-  -D-glucopyranoside
(GlcNAc-Ins) . A transposon mutant of Mycobacterium smegmatis
shown to be GlcNAc-Ins and mycothiol deficient was sequenced to
identify a putative glycosyltransferase gene designated mshA .
The ortholog in Mycobacterium tuberculosis, Rv0486, was used
to complement the mutant phenotype .
Use of Bromodeoxyuridine Immunocapture To Identify Active Bacteria Associated with Arbuscular Mycorrhizal Hyphae. Veronica Artursson, 2003.Arbuscular mycorrhizae are beneficial for crops grown under low-till management systems . Increasingly, it is becoming apparent that bacteria associated with mycorrhizae can enhance the beneficial relationship between mycorrhizae and plants . However, it has been difficult to study these relationships by conventional techniques . In this study actively growing bacteria were identified in soil from an undisturbed fallow field known to contain arbuscular mycorrhizae by using molecular tools to eliminate the need for cultivation . A thymidine analog, bromodeoxyuridine (BrdU), was added to the soil and incubated for 2 days . DNA was extracted, and the newly synthesized DNA was isolated by immunocapture of the BrdU-containing DNA . The active bacteria in the community were identified by 16S rRNA gene PCR amplification and DNA sequence analysis . Based on 16S rRNA gene sequence information, a selective medium was chosen to isolate the corresponding active bacteria . Bacillus cereus strain VA1, one of the bacteria identified by the BrdU method, was isolated from the soil and tagged with green fluorescent protein . By using confocal microscopy, this bacterium was shown to clearly attach to arbuscular mycorrhizal hyphae . This study was the first to use this combination of molecular and traditional approaches to isolate, identify, and visualize a specific bacterium that is active in fallow soil and associates with arbuscular mycorrhizal hyphae .
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