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Purification and Properties of an Intracellular 3-Hydroxybutyrate-Oligomer Hydrolase (PhaZ2) in Ralstonia eutropha H16 and Its Identification as a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase. Teruyuki Kobayashi, 2003.An intracellular 3-hydroxybutyrate (3HB)-oligomer hydrolase (PhaZ2Reu) of Ralstonia eutropha was purified from Escherichia coli harboring a plasmid containing phaZ2Reu . The purified enzyme hydrolyzed linear and cyclic 3HB-oligomers . Although it did not degrade crystalline poly(3-hydroxybutyrate) (PHB), the purified enzyme degraded artificial amorphous PHB at a rate similar to that of the previously identified intracellular PHB (iPHB) depolymerase (PhaZ1Reu) . The enzyme appeared to be an endo-type hydrolase, since it actively hydrolyzed cyclic 3HB-oligomers . However, it degraded various linear 3HB-oligomers and amorphous PHB in the fashion of an exo-type hydrolase, releasing one monomer unit at a time . PhaZ2 was found to bind to PHB inclusion bodies and as a soluble enzyme to cell-free supernatant fractions in R . eutropha; in contrast, PhaZ1 bound exclusively to the inclusion bodies . When R . eutropha H16 was cultivated in a nutrient-rich medium, the transient deposition of PHB was observed: the content of PHB was maximized in the log growth phase (12 h, ca . 14% PHB of dry cell weight) and decreased to a very low level in the stationary phase (ca . 1% of dry cell weight) . In each phaZ1-null mutant and phaZ2-null mutant, the PHB content in the cell increased to ca . 5% in the stationary phase . A double mutant lacking both phaZ1 and phaZ2 showed increased PHB content in the log phase (ca . 20%) and also an elevated PHB level (ca . 8%) in the stationary phase . These results indicate that PhaZ2 is a novel iPHB depolymerase, which participates in the mobilization of PHB in R . eutropha along with PhaZ1 . Ketolide Antimicrobial Activity Persists after Disruption of Interactions with Domain II of 23S rRNA. Guy W. Novotny, 2004.Ketolides are the latest derivatives developed from the macrolide erythromycin to improve antimicrobial activity . All macrolides and ketolides bind to the 50S ribosomal subunit, where they come into contact with adenosine 2058 (A2058) within domain V of the 23S rRNA and block protein synthesis . An additional interaction at nucleotide A752 in the rRNA domain II is made via the synthetic carbamate-alkyl-aryl substituent in the ketolides HMR3647 (telithromycin) and HMR3004, and this interaction contributes to their improved activities . Only a few macrolides, including tylosin, come into contact with domain II of the rRNA and do so via interactions with nucleotides G748 and A752 . We have disrupted these macrolide-ketolide interaction sites in the rRNA to assess their relative importance for binding . Base substitutions at A752 were shown to confer low levels of resistance to telithromycin but not to HMR3004, while deletion of A752 confers low levels of resistance to both ketolides . Mutations at position 748 confer no resistance . Substitution of guanine at A2058 gives rise to the MLSB (macrolide, lincosamide, and streptogramin B) phenotype, which confers resistance to all the drugs . However, resistance to ketolides was abolished when the mutation at position 2058 was combined with a mutation in domain II of the same rRNA . In contrast, the same dual mutations in rRNAs conferred enhanced resistance to tylosin . Our results show that the domain II interactions of telithromycin and HMR3004 differ from each other and from those of tylosin . The data provide no indication that mutations within domain II, either alone or in combination with an A2058 mutation, can confer significant levels of telithromycin resistance . Pharmacokinetics of Intravenous Itraconazole in Stable Hemodialysis Patients. John F. Mohr, 2004.The pharmacokinetics of intravenous itraconazole (ITC) was studied in dialysis patients . Dialysis had no effect on the half-life and clearance of ITC or OH-ITC . However, dialysis allowed the clearance of hydroxypropyl-ß-cyclodextrin (HP-ß-CD) . The area under the concentration-time curve from time zero to infinity (AUC0-  ) for HP-ß-CD administered before dialysis was lower than the AUC0- when it was administered after dialysis (P < 0.01) . Administration of ITC intravenously just prior to hemodialysis appears to produce adequate systemic exposures of ITC and OH-ITC while allowing dialysis clearance of HP-ß-CD . Studies of multiple administrations are warranted .
Characterization of the Two-Component Abortive Phage Infection Mechanism AbiT from Lactococcus lactis. Julie D. Bouchard, 2002.During the production of fermented dairy products, virulent bacteriophages infecting Lactococcus lactis can delay or stop the milk acidification process . A solution to this biological problem consists of introducing natural phage barriers into the strains used by the dairy industry . One such hurdle is called abortive infection (Abi) and causes premature cell death with no or little phage progeny . Here, we describe the isolation and characterization of a novel Abi mechanism encoded by plasmid pED1 from L . lactis . The system is composed of two constitutively cotranscribed genes encoding putative proteins of 127 and 213 amino acids, named AbiTi and AbiTii, respectively . Site-directed mutagenesis indicated that a hydrophobic region at the C-terminal extremity of AbiTi is essential to the antiphage phenotype . The AbiT system is effective against phages of the 936 and P335 species (efficiency of plaquing between 10-5 and 10-7) and causes a 20-fold reduction in the efficiency to form centers of infection as well as a 10- to 12-fold reduction in the burst size . Its efficacy could be improved by raising the plasmid copy number, but changing the intrinsic ratio of AbiTi and AbiTii did not greatly affect the antiphage activity . The monitoring of the intracellular phage infection process by DNA replication, gene expression, and electron microscopy as well as the study of phage mutants by genome mapping indicated that AbiT is likely to act at a later stage of the phage lytic cycle . Translocated Intimin Receptor and Its Chaperone Interact with ATPase of the Type III Secretion Apparatus of Enteropathogenic Escherichia coli. Annick Gauthier, 2003.Few interactions have been reported between effectors and components of the type III secretion apparatus, although many interactions have been demonstrated between type III effectors and their cognate chaperones . It is thought that chaperones may play a role in directing effectors to the type III secretion apparatus . The ATPase FliI in the flagellar assembly apparatus plays a pivotal role in interacting with other components of the apparatus and with substrates of the flagellar system . We performed experiments to determine if there were any interactions between the effector Tir and its chaperone CesT and the type III secretion apparatus of enteropathogenic Escherichia coli (EPEC) . Specifically, based on analogies with the flagella system, we examined Tir-CesT interactions with the putative ATPase EscN . We showed by affinity chromatography that EscN and Tir bind CesT specifically . Tir is not necessary for CesT and EscN interactions, and EscN binds Tir specifically without its chaperone CesT . Moreover, Tir directly binds EscN, as shown via gel overlay and enzyme-linked immunosorbent assay, and coimmunoprecipitation experiments revealed that Tir interacts with EscN inside EPEC . These data provide evidence for direct interactions between a chaperone, effector, and type III component in the pathogenic type III secretion system and suggest a model for Tir translocation whereby its chaperone, CesT, brings Tir to the type III secretion apparatus by specifically interacting with the type III ATPase EscN . Molecular Characterization of Sulfate-Reducing Bacteria in the Guaymas Basin. Ashita Dhillon, 2003.The Guaymas Basin (Gulf of California) is a hydrothermal vent site where thermal alteration of deposited planktonic and terrestrial organic matter forms petroliferous material which supports diverse sulfate-reducing bacteria . We explored the phylogenetic and functional diversity of the sulfate-reducing bacteria by characterizing PCR-amplified dissimilatory sulfite reductase (dsrAB) and 16S rRNA genes from the upper 4 cm of the Guaymas sediment . The dsrAB sequences revealed that there was a major clade closely related to the acetate-oxidizing delta-proteobacterial genus Desulfobacter and a clade of novel, deeply branching dsr sequences related to environmental dsr sequences from marine sediments in Aarhus Bay and Kysing Fjord (Denmark) . Other dsr clones were affiliated with gram-positive thermophilic sulfate reducers (genus Desulfotomaculum) and the delta-proteobacterial species Desulforhabdus amnigena and Thermodesulforhabdus norvegica . Phylogenetic analysis of 16S rRNAs from the same environmental samples resulted in identification of four clones affiliated with Desulfobacterium niacini, a member of the acetate-oxidizing, nutritionally versatile genus Desulfobacterium, and one clone related to Desulfobacula toluolica and Desulfotignum balticum . Other bacterial 16S rRNA bacterial phylotypes were represented by non-sulfate reducers and uncultured lineages with unknown physiology, like OP9, OP8, as well as a group with no clear affiliation . In summary, analyses of both 16S rRNA and dsrAB clone libraries resulted in identification of members of the Desulfobacteriales in the Guaymas sediments . In addition, the dsrAB sequencing approach revealed a novel group of sulfate-reducing prokaryotes that could not be identified by 16S rRNA sequencing .
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