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The Homogentisate Pathway: a Central Catabolic Pathway Involved in the Degradation of L-Phenylalanine, L-Tyrosine, and 3-Hydroxyphenylacetate in Pseudomonas putida. Elsa Arias-Barrau, 2004.Pseudomonas putida metabolizes Phe and Tyr through a peripheral pathway involving hydroxylation of Phe to Tyr (PhhAB), conversion of Tyr into 4-hydroxyphenylpyruvate (TyrB), and formation of homogentisate (Hpd) as the central intermediate . Homogentisate is then catabolized by a central catabolic pathway that involves three enzymes, homogentisate dioxygenase (HmgA), fumarylacetoacetate hydrolase (HmgB), and maleylacetoacetate isomerase (HmgC), finally yielding fumarate and acetoacetate . Whereas the phh, tyr, and hpd genes are not linked in the P . putida genome, the hmgABC genes appear to form a single transcriptional unit . Gel retardation assays and lacZ translational fusion experiments have shown that hmgR encodes a specific repressor that controls the inducible expression of the divergently transcribed hmgABC catabolic genes, and homogentisate is the inducer molecule . Footprinting analysis revealed that HmgR protects a region in the Phmg promoter that spans a 17-bp palindromic motif and an external direct repetition from position –16 to position 29 with respect to the transcription start site . The HmgR protein is thus the first IclR-type regulator that acts as a repressor of an aromatic catabolic pathway . We engineered a broad-host-range mobilizable catabolic cassette harboring the hmgABC, hpd, and tyrB genes that allows heterologous bacteria to use Tyr as a unique carbon and energy source . Remarkably, we show here that the catabolism of 3-hydroxyphenylacetate in P . putida U funnels also into the homogentisate central pathway, revealing that the hmg cluster is a key catabolic trait for biodegradation of a small number of aromatic compounds . Randomized Comparison of Serum Teicoplanin Concentrations following Daily or Alternate Daily Dosing in Healthy Adults. Bernard Rouveix, 2004.Trough serum teicoplanin concentrations were compared in healthy adults following intravenous administration of one of two regimens: (i) 12 mg/kg of body weight every 12 h for 3 doses and then 15 mg/kg every 48 h for 4 doses (n = 16 subjects) or (ii) 6 mg/kg every 12 h for 2 doses and then 6 mg/kg every 24 h for 9 doses (n = 8 subjects) . The mean ± standard deviation trough concentrations in serum on day 11 (24 and 48 h after administration of the last dose for the daily and alternate-day dosing schedules, respectively) were 16.0 ± 2.1 and 17.9 ± 3.5 mg/liter for subjects receiving the two regimens, respectively, by a fluorescence polarization immunoassay . The limits of the 95% confidence interval of the difference (–0.2, 3.6 mg/liter) determined by a nonparametric test were situated above the –1.3-mg/liter maximum set difference and indicated a noninferiority of the alternate-day dosing to the daily dosing . Throughout the study the individual trough concentrations in serum in the alternate-day dosing group constantly exceeded 10 mg/liter, the presently recommended target concentration for the treatment of severe infections . The trough concentrations in the sera of all subjects were bactericidal for six Staphylococcus aureus strains for which teicoplanin MICs are between 0.5 and 4 mg/liter . The bactericidal activity of serum was related to total teicoplanin (protein bound and unbound) . In conclusion, an alternate-day dosing schedule (15 mg/kg on alternate days following administration of a 12-mg/kg loading dose three times every 12 h) could be considered for further efficacy and safety studies . The Membrane-Associated Protein-Serine/Threonine Kinase from Sulfolobus solfataricus Is a Glycoprotein. Brian H. Lower, 2002.Treatment of a sodium dodecyl sulfate-polyacrylamide gel with periodic acid-Schiff (PAS) stain or blotting with Galanthus nivalis agglutinin revealed the presence of several glycosylated polypeptides in a partially purified detergent extract of the membrane fraction of Sulfolobus solfataricus. One of the glycoproteins comigrated with the membrane-associated protein-serine/threonine kinase from S . solfataricus, which had been radiolabeled by autophosphorylation with [32P]ATP in vitro . Treatment with a chemical deglycosylating agent, trifluoromethanesulfonic acid, abolished PAS staining and reduced the Mr of the protein kinase from  67,000 to
62,000 . Protein kinase activity also adhered to, and could be eluted from, agarose beads containing bound G . nivalis agglutinin . Glycosylation of the protein kinase implies that at least a portion of this integral membrane protein resides on the external surface of the cell membrane .
In Vivo DNA-Binding and Oligomerization Properties of the Shigella flexneri AraC-Like Transcriptional Regulator VirF as Identified by Random and Site-Specific Mutagenesis. Megan E. Porter, 2002.In Shigella flexneri expression of the plasmid-encoded virulence genes is regulated via a complex mechanism involving both environmental signals and specific transactivators . The primary regulator protein, VirF, is a member of the AraC family of transcription factors and shares with other AraC-like proteins a conserved carboxy-terminal domain thought to be important for DNA binding . Random and site-directed mutagenesis of the virF gene encoding VirF yielded a number of mutations along the length of the protein which severely affected the ability of VirF to activate gene expression . The mutant proteins were shown to be affected in their ability to activate the virulence genes virB and icsA, both known to be regulated directly by VirF, as well as the virB-dependent virulence gene mxiC . Mutating key residues predicted to be important for DNA recognition had a significant negative effect, thereby suggesting that VirF interacts with its target sequence via two helix-turn-helix motifs . Two mutants that were dominant negative when coexpressed with the wild-type VirF protein were also isolated, indicating a role for protein-protein oligomerization in normal VirF function . mRNA Extraction and Reverse Transcription-PCR Protocol for Detection of nifH Gene Expression by Azotobacter vinelandii in Soil. Helmut Bürgmann, 2003.The study of free-living nitrogen-fixing organisms in bulk soil is hampered by the great diversity of soil microbial communities and the difficulty of relating nitrogen fixation activities to individual members of the diazotroph populations . We developed a molecular method that allows analysis of nifH mRNA expression in soil in parallel with determinations of nitrogen-fixing activity and bacterial growth . In this study, Azotobacter vinelandii growing in sterile soil and liquid culture served as a model system for nifH expression, in which sucrose served as the carbon source and provided nitrogen-limited conditions, while amendments of NH4NO3 were used to suppress nitrogen fixation . Soil RNA extraction was performed with a new optimized direct extraction protocol that yielded nondegraded total RNA . The RNA extracts were of high purity, free of DNA contamination, and allowed highly sensitive and specific detection of nifH mRNA by a reverse transcription-PCR . The level of nifH gene expression was estimated by PCR amplification of reverse-transcribed nifH mRNA fragments with A . vinelandii-specific nifH primers . This new approach revealed that nifH gene expression was positively correlated with bulk nitrogen fixation activity in soil (r2 = 0.72) and in liquid culture (r2 = 0.84) and therefore is a powerful tool for studying specific regulation of gene expression directly in the soil environment .
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