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Nonisotopic, Semiautomated Plasmodium falciparum Bioassay for Measurement of Antimalarial Drug Levels in Serum or Plasma. Harald Noedl, 2004.A simple, nonisotopic, semiautomated bioassay for the measurement of antimalarial drug levels in plasma or serum based on the quantitation of histidine-rich protein II in malaria culture is presented . The assay requires only small sample volumes and was found to be highly sensitive and reproducible . The results closely paralleled those obtained with isotopic bioassays (R = 0.988, P < 0.001) and high-performance liquid chromatography-electrochemical detection (R = 0.978, P < 0.001) . N-Acyl-Homoserine Lactone Inhibition of Rhizobial Growth Is Mediated by Two Quorum-Sensing Genes That Regulate Plasmid Transfer. A. Wilkinson, 2002.The growth of some strains of Rhizobium leguminosarum bv . viciae is inhibited by N-(3-hydroxy-7-cis tetradecenoyl)-L-homoserine lactone (3OH-C14:1-HSL), which was previously known as the small bacteriocin before its characterization as an N-acyl homoserine lactone (AHL) . Tn5-induced mutants of R . leguminosarum bv . viciae resistant to 3OH-C14:1-HSL were isolated, and mutations in two genes were identified . These genes, bisR and triR, which both encode LuxR-type regulators required for plasmid transfer, were found downstream of an operon containing trb genes involved in the transfer of the symbiotic plasmid pRL1JI . The first gene in this operon is traI, which encodes an AHL synthase, and the trbBCDEJKLFGHI genes were found between traI and bisR . Mutations in bisR, triR, traI, or trbL blocked plasmid transfer . Using gene fusions, it was demonstrated that bisR regulates triR in response to the presence of 3OH-C14:1-HSL . In turn, triR is then required for the induction of the traI-trb operon required for plasmid transfer . bisR also represses expression of cinI, which is chromosomally located and determines the level of production of 3OH-C14:1-HSL . The cloned bisR and triR genes conferred 3OH-C14:1-HSL sensitivity to strains of R . leguminosarum bv . viciae normally resistant to this AHL . Furthermore, bisR and triR made Agrobacterium tumefaciens sensitive to R . leguminosarum bv . viciae strains producing 3OH-C14:1-HSL . Analysis of patterns of growth inhibition using mutant strains and synthetic AHLs revealed that maximal growth inhibition required, in addition to 3OH-C14:1-HSL, the presence of other AHLs such as N-octanoyl-L-homoserine lactone and/or N-(3-oxo-octanoyl)-L-homoserine lactone . In an attempt to identify the causes of growth inhibition, a strain of R . leguminosarum bv . viciae carrying cloned bisR and triR was treated with an AHL extract containing 3OH-C14:1-HSL . N-terminal sequencing of induced proteins revealed one with significant similarity to the protein translation factor Ef-Ts . Characterization of GlnK1 from Methanosarcina mazei Strain Gö1: Complementation of an Escherichia coli glnK Mutant Strain by GlnK1. Claudia Ehlers, 2002.Trimeric PII-like signal proteins are known to be involved in bacterial regulation of ammonium assimilation and nitrogen fixation . We report here the first biochemical characterization of an archaeal GlnK protein from the diazotrophic methanogenic archaeon Methanosarcina mazei strain Gö1 and show that M . mazei GlnK1 is able to functionally complement an Escherichia coli glnK mutant for growth on arginine . This indicates that the archaeal GlnK protein substitutes for the regulatory function of E . coli GlnK . M . mazei GlnK1 is encoded in the glnK1-amtB1 operon, which is transcriptionally regulated by the availability of combined nitrogen and is only transcribed in the absence of ammonium . The deduced amino acid sequence of the archaeal glnK1 shows 44% identity to the E . coli GlnK and contains the conserved tyrosine residue (Tyr-51) in the T-loop structure . M . mazei glnK1 was cloned and overexpressed in E . coli, and GlnK1 was purified to apparent homogeneity . A molecular mass of 42 kDa was observed under native conditions, indicating that its native form is a trimer . GlnK1-specific antibodies were raised and used to confirm the in vivo trimeric form by Western analysis . In vivo ammonium upshift experiments and analysis of purified GlnK1 indicated significant differences compared to E . coli GlnK . First, GlnK1 from M . mazei is not covalently modified by uridylylation under nitrogen limitation . Second, heterotrimers between M . mazei GlnK1 and Klebsiella pneumoniae GlnK are not formed . Because M . mazei GlnK1 was able to complement growth of an E . coli glnK mutant with arginine as the sole nitrogen source, it is likely that uridylylation is not required for its regulatory function . Pilin-Like Proteins in the Extremely Thermophilic Bacterium Thermus thermophilus HB27: Implication in Competence for Natural Transformation and Links to Type IV Pilus Biogenesis. Alexandra Friedrich, 2003.The extreme thermophile Thermus thermophilus HB27 exhibits high frequencies of natural transformation . Although we recently reported identification of the first competence genes in Thermus, the molecular basis of DNA uptake is unknown . A pilus-like structure is assumed to be involved . Twelve genes encoding prepilin-like proteins were identified in three loci in the genome of T . thermophilus . Mutational analyses, described in this paper, revealed that one locus, which contains four genes that encode prepilin-like proteins (pilA1 to pilA4), is essential for natural transformation . Additionally, comZ, a new competence gene with no similarity to known genes, was identified . Analysis of the piliation phenotype revealed wild-type piliation of a pilA1-pilA3  kat mutant and a comZ mutant, whereas a pilA4 mutant was found to be completely devoid of pilus structures . These findings, together with the significant similarity of PilA4 to prepilins, led to the conclusion that the T . thermophilus pilus structures are type IV pili . Furthermore, the loss of the transformation and piliation phenotype in the pilA4 mutant suggests that type IV pili are implicated in natural transformation of T . thermophilus HB27 .
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