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High-Resolution Structure of the Histidine-Containing Phosphocarrier Protein (HPr) from Staphylococcus aureus and Characterization of Its Interaction with the Bifunctional HPr Kinase/Phosphorylase. Till Maurer, 2004.A high-resolution structure of the histidine-containing phosphocarrier protein (HPr) from Staphylococcus aureus was obtained by heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopy on the basis of 1,766 structural restraints . Twenty-three hydrogen bonds in HPr could be directly detected by polarization transfer from the amide nitrogen to the carbonyl carbon involved in the hydrogen bond . Differential line broadening was used to characterize the interaction of HPr with the HPr kinase/phosphorylase (HPrK/P) of Staphylococcus xylosus, which is responsible for phosphorylation-dephosphorylation of the hydroxyl group of the regulatory serine residue at position 46 . The dissociation constant Kd was determined to be 0.10 ± 0.02 mM at 303 K from the NMR data, assuming independent binding . The data are consistent with a stoichiometry of 1 HPr molecule per HPrK/P monomer in solution . Using transversal relaxation optimized spectroscopy-heteronuclear single quantum correlation, we mapped the interaction site of the two proteins in the 330-kDa complex . As expected, it covers the region around Ser46 and the small helix b following this residue . In addition, HPrK/P also binds to the second phosphorylation site of HPr at position 15 . This interaction may be essential for the recognition of the phosphorylation state of His15 and the phosphorylation-dependent regulation of the kinase/phosphorylase activity . In accordance with this observation, the recently published X-ray structure of the HPr/HPrK core protein complex from Lactobacillus casei shows interactions with the two phosphorylation sites . However, the NMR data also suggest differences for the full-length protein from S . xylosus: there are no indications for an interaction with the residues preceding the regulatory Ser46 residue (Thr41 to Lys45) in the protein of S . xylosus . In contrast, it seems to interact with the C-terminal helix of HPr in solution, an interaction which is not observed for the complex of HPr with the core of HPrK/P of L . casei in crystals . Open Reading Frame sso2387 from the Archaeon Sulfolobus solfataricus Encodes a Polypeptide with Protein-Serine Kinase Activity. Brian H. Lower, 2003.The predicted polypeptide product of open reading frame sso2387 from the archaeon Sulfolobus solfataricus, SsoPK2, displayed several of the sequence features conserved among the members of the "eukaryotic" protein kinase superfamily . sso2387 was cloned, and its polypeptide product was expressed in Escherichia coli . The recombinant protein, rSsoPK2, was recovered in insoluble aggregates that could be dispersed by using high concentrations (5 M) of urea . The solubilized polypeptide displayed the ability to phosphorylate itself as well as several exogenous proteins, including mixed histones, casein, bovine serum albumin, and reduced carboxyamidomethylated and maleylated lysozyme, on serine residues . The source of this activity resided in that portion of the protein displaying homology to the catalytic domain of eukaryotic protein kinases . By use of mass spectrometry, the sites of autophosphorylation were found to be located in two areas, one immediately N terminal to the region corresponding to subdomain I of eukaryotic protein kinases, and the second N terminal to the presumed activation loop located between subdomains VII and VIII . Autophosphorylation of rSsoPK2 could be uncoupled from the phosphorylation of exogenous proteins by manipulation of the temperature or mutagenic alteration of the enzyme . Autophosphorylation was detected only at temperatures  60°C,
whereas phosphorylation of exogenous proteins was detectable at 37°C .
Similarly, replacement of one of the potential sites of
autophosphorylation, Ser548, with alanine blocked
autophosphorylation but not phosphorylation of an exogenous protein,
casein .
Pharmacodynamics of Pulse Dosing versus Standard Dosing: In Vitro Metronidazole Activity against Bacteroides fragilis and Bacteroides thetaiotaomicron. Khalid H. Ibrahim, 2004.Pulse dosing is a novel approach to dosing that produces escalating antibiotic levels early in the dosing interval followed by a prolonged dose-free period . Antibiotic is frontloaded by means of four sequential bolus injections, after which antibiotic levels are allowed to diminish until the next dose . This study compares standard thrice-daily dosing and pulse dosing of metronidazole against Bacteroides spp . in an in vitro model . Two American Type Culture Collection Bacteroides fragilis isolates (metronidazole MIC for each organism = 1 mg/liter) were exposed to metronidazole for 48 or 96 h . Human pharmacokinetics were simulated for an oral 500-mg dose given every 8 h (maximum concentration of drug [Cmax] = 12 mg/liter; half-life = 8 h; area under the curve [AUC] = 294 mg · h/liter) and for pulse dosing . Pulses, each producing an increase in metronidazole concentration of 9 mg/liter, were administered at times 0, 2, 4, and 6 h of each 24-h cycle, with a targeted half-life of 8 h (AUC = 347 mg · h/liter) . A metronidazole-resistant B . fragilis strain (metronidazole MIC = 32 mg/liter) was exposed to both dosing regimens and, additionally, to a regimen of 1,500 mg administered once daily (Cmax = 36 mg/liter; AUC = 364 mg · h/liter) . Furthermore, regimens against one B . fragilis isolate and one B . thetaiotaomicron isolate corresponding to one-fourth and one-eighth of the thrice-daily and pulse dosing regimens, mimicking peak metronidazole concentrations achieved in abscesses, were simulated in 48-h experiments (metronidazole MIC = 1 mg/liter) . Time-kill curves were generated for each experiment and analyzed for bactericidal activity, defined as a bacterial burden reduction  3 log10 CFU/ml . The results of paired (Wilcoxon matched-pair signed-rank test) and nonpaired (Mann-Whitney test) statistical analyses conducted on time to 3 log10 kill data and area under the kill curve data from each of the thrice-daily dosing experiments versus each of the pulse dosing experiments were considered not significant for a given isolate-dosing regimen combination . The thrice-daily dosing, pulse dosing, and once-daily dosing regimens all exhibited bactericidal activity . Metronidazole administered in standard or pulse dosing fashion was highly active against both susceptible and resistant strains of Bacteroides spp .
Identification and Localization of the Gene Cluster Encoding Biosynthesis of the Antitumor Macrolactam Leinamycin in Streptomyces atroolivaceus S-140. Yi-Qiang Cheng, 2002. Genetic Variation at the vlsE Locus of Borrelia burgdorferi within Ticks and Mice over the Course of a Single Transmission Cycle. Jun Ohnishi, 2003.The Lyme disease spirochete, Borrelia burgdorferi, causes a persistent infection in the vertebrate host even though infected animals mount an active immune response against the spirochete . One strategy used by the spirochete to evade vertebrate host immunity is to vary the structure and expression of outer membrane antigens . The vlsE locus represents the best-studied example of antigenic variation in B . burgdorferi . During vertebrate host infection, recombination between the active vlsE locus and silent, partial vlsE copies leads to gene conversion events and the generation of novel alleles at the expression site . In the present study, we followed a population of B . burgdorferi organisms moving through vertebrate host and tick stages to complete one transmission cycle . The major goal of the study was to determine if the vlsE locus was subject to different selective pressure and/or recombination frequency at different stages of the spirochete's life cycle . We report here that the vlsE genetic diversity generated within the rodent host was maintained through the larval and nymphal tick stages . Therefore, naturally infected ticks are likely to transmit spirochete populations with multiple vlsE alleles into naive vertebrate hosts . Although vlsE genetic diversity in mice was maintained through tick stages, the dominant vlsE alleles were different between tick stages as well as between individual ticks . We propose that population-level bottlenecks experienced by spirochetes, especially during the larval-to-nymphal molt, are responsible for individual infected ticks harboring different dominant vlsE alleles . Although vlsE genetic diversity is maintained through tick stages, the VlsE protein is unlikely to be of functional importance in the vector, because the protein was expressed by very few (<1%) bacteria in the vector . Determination of the Domain of the Lactobacillus delbrueckii subsp . bulgaricus Cell Surface Proteinase PrtB Involved in Attachment to the Cell Wall after Heterologous Expression of the prtB Gene in Lactococcus lactis. Jacques-Edouard Germond, 2003.Belonging to the subtilase family, the cell surface proteinase (CSP) PrtB of Lactobacillus delbrueckii subsp . bulgaricus differs from other CSPs synthesized by lactic acid bacteria . Expression of the prtB gene under its own promoter was shown to complement the proteinase-deficient strain MG1363 (PrtP- PrtM-) of Lactococcus lactis subsp . cremoris . Surprisingly, the maturation process of PrtB, unlike that of lactococcal CSP PrtPs, does not require a specific PrtM-like chaperone . The carboxy end of PrtB was previously shown to be different from the consensus anchoring region of other CSPs and exhibits an imperfect duplication of 59 amino acids with a high lysine content . By using a deletion strategy, the removal of the last 99 amino acids, including the degenerated anchoring signal (LPKKT), was found to be sufficient to release a part of the truncated PrtB into the culture medium and led to an increase in PrtB activity . This truncated PrtB is still active and enables L . lactis MG1363 to grow in milk supplemented with glucose . By contrast, deletion of the last 806 amino acids of PrtB led to the secretion of an inactive proteinase . Thus, the utmost carboxy end of PrtB is involved in attachment to the bacterial cell wall . Proteinase PrtB constitutes a powerful tool for cell surface display of heterologous proteins like antigens .
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