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Role of Histone-Like Protein H-NS in Multidrug Resistance of Escherichia coli. Kunihiko Nishino, 2004.The histone-like protein H-NS is a major component of the bacterial nucleoid and plays a crucial role in global gene regulationof enteric bacteria . It is known that the expression of a varietyof genes is repressed by H-NS, and mutations in hns result in various phenotypes, but the role of H-NS in the drug resistanceof Escherichia coli has not been known . Here we present datashowing that H-NS contributes to multidrug resistance by regulatingthe expression of multidrug exporter genes . Deletion of thehns gene from the deltaacrAB mutant increased levels of resistanceagainst antibiotics, antiseptics, dyes, and detergents . Decreasedaccumulation of ethidium bromide and rhodamine 6G in the hnsmutant compared to that in the parental strain was observed,suggesting the increased expression of some drug exporter[s]in this mutant . The increased drug resistance and decreaseddrug accumulation caused by the hns deletion were completelysuppressed by deletion of the multifunctional outer membranechannel gene tolC . At least eight drug exporter systems requireTolC for their functions . Among these, increased expressionof acrEF, mdtEF, and emrKY was observed in the deltahns strain byquantitative real-time reverse transcription-PCR analysis . The deltahns-mediated multidrug resistance pattern is quite similar tothat caused by overproduction of the AcrEF exporter . Deletionof the acrEF gene greatly suppressed the level of deltahns-mediated multidrug resistance . However, this strain still retained resistance to some compounds . The remainder of the multidrug resistance pattern was similar to that conferred by overproduction of theMdtEF exporter . Double deletion of the mdtEF and acrEF genes completely suppressed deltahns-mediated multidrug resistance, indicatingthat deltahns-mediated multidrug resistance is due to derepressionof the acrEF and mdtEF drug exporter genes. Optimization of Procedures for Isolation of Mycobacteria from Soil and Water Samples Obtained in Northern India. Deepti Parashar, 2004.For isolation of environmental mycobacteria, a decontamination procedure has been standardized by which treatment with 3% sodium dodecyl sulfate plus 4% NaOH (15 and 30 min for rapid and slow growers, respectively) is followed by incubation with 2% cetrimide (5 and 15 min for fast- and slow-growing mycobacteria, respectively); this procedure was found to completely eliminate contamination with other organisms and resulted in the isolation of only mycobacteria . Up-Regulation of the Yersinia enterocolitica yop Regulon by Deletion of the Flagellum Master Operon flhDC. Sophie Bleves, 2002.The Yop virulon enables extracellularly located Yersinia, in close contact with a eukaryotic target cell, to inject bacterial toxic proteins directly into the cytosol of this cell . Several Ysc proteins, forming the Yop secretion apparatus, display homology with proteins of the flagellar basal body . To determine whether this relationship could extend to the regulatory pathways, we analyzed the influence of flhDC, the master regulatory operon of the flagellum, on the yop regulon . In an flhDC mutant, the yop regulon was up-regulated . The transcription of virF and the steady-state level of the transcriptional activator VirF were enhanced . yop transcription was increased at 37°C and could also be detected at a low temperature . Yop secretion was increased at 37°C and occurred even at a low temperature . The Ysc secretion machinery was thus functional at room temperature in the absence of flagella, implying that in wild-type bacteria, FlhD and/or FlhC, or the product of a gene downstream of flhDC, represses the yop regulon . In agreement with this notion, increased expression of flhDC in wild-type bacteria resulted in the oversecretion of flagellins at room temperature and in decreased Yop secretion at 37°C . Expression of magA in Legionella pneumophila Philadelphia-1 Is Developmentally Regulated and a Marker of Formation of Mature Intracellular Forms. Margot F. Hiltz, 2004.Legionella pneumophila displays a biphasic developmental cycle in which replicating forms (RFs) differentiate postexponentially into highly infectious, cyst-like mature intracellular forms (MIFs) . Using comparative protein profile analyses (MIFs versus RFs), we identified a 20-kDa protein, previously annotated as "Mip-like" protein, that was enriched in MIFs . However, this 20-kDa protein shared no similarity with Mip, a well-characterized peptidyl-prolyl isomerase of L . pneumophila, and for clarity we renamed it MagA (for "MIF-associated gene") . We monitored MagA levels across the growth cycle (in vitro and in vivo) by immunoblotting and established that MagA levels increased postexponentially in vitro (  3-fold) and nearly 10-fold during MIF morphogenesis in HeLa cells . DNA sequence analysis of the magA locus revealed an upstream divergently transcribed gene, msrA, encoding a peptide methionine sulfoxide reductase and a shared promoter region containing direct and indirect repeat sequences as well as –10 hexamers often associated with stationary-phase regulation . While MagA has no known function, it contains a conserved CXXC motif commonly found in members of the thioredoxin reductase family and in AhpD reductases that are associated with alkylhydroperoxide reductase (AhpC), suggesting a possible role in protection from oxidative stress . MIFs from L . pneumophila strain Lp02 containing a magA deletion exhibited differences in Giménez staining, as well as an apparent increase in cytopathology to HeLa cells, but otherwise were unaltered in virulence traits . As demonstrated by this study, MagA appears to be a MIF-specific protein expressed late in intracellular growth that may serve as a useful marker of development .
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