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FlhF, the Third Signal Recognition Particle-GTPase of Bacillus subtilis, Is Dispensable for Protein Secretion. Geeske Zanen, 2004.Bacillus subtilis contains three proteins of the signal recognition particle-GTPase family known as Ffh, FtsY, and FlhF . Here we show that FlhF is dispensable for protein secretion, unlike Ffh and FtsY . Although flhF is located in the fla/che operon, B . subtilis 168 flhF mutant cells assemble flagella and are motile . Dose-Dependent Resorption of Quinine after Intrarectal Administration to Children with Moderate Plasmodium falciparum Malaria. Eric Pussard, 2004.The pharmacokinetics of increasing doses of an intrarectal Cinchona alkaloid combination containing 96.1% quinine, 2.5% quinidine, 0.68% cinchonine, and 0.67% cinchonidine (Quinimax) was compared to that of parenteral regimens in 60 children with moderate malaria . Quinine exhibited a nonlinear pharmacokinetics, suggesting a saturation of rectal resorption . When early rejections appeared, blood quinine concentrations decreased by 30 to 50% and were restored by an immediate half-dose administration of the drug . Rectal administration of doses of 16 or 20 mg/kg of body weight led to concentration-time profiles in blood similar to those of parenteral regimens and could be an early treatment of childhood malaria . Characterization of Plasmid pOR1 from Ornithobacterium rhinotracheale and Construction of a Shuttle Plasmid. Ruud Jansen, 2004.The bacterium Ornithobacterium rhinotracheale has been recognized as an emerging pathogen in poultry since about 10 years ago . Knowledge of this bacterium and its mechanisms of virulence is still very limited . Here we report the development of a transformation system that enables genetic modification of O . rhinotracheale . The system is based on a cryptic plasmid, pOR1, that was derived from an O . rhinotracheale strain of serotype K . Sequencing indicated that the plasmid consisted of 14,787 nucleotides . Sequence analysis revealed one replication origin and several rep genes that control plasmid replication and copy number, respectively . In addition, pOR1 contains genes with similarity to a heavy-metal-transporting ATPase, a TonB-linked siderophore receptor, and a laccase . Reverse transcription-PCR demonstrated that these genes were transcribed . Other putative open reading frames exhibited similarities with a virulence-associated protein in Actinobacillus actinomycetemcomitans and a number of genes coding for proteins with unknown function . An Escherichia coli-O . rhinotracheale shuttle plasmid (pOREC1) was constructed by cloning the replication origin and rep genes from pOR1 and the cfxA gene from Bacteroides vulgatus, which codes for resistance to the antibiotic cefoxitin, into plasmid pGEM7 by using E . coli as a host . pOREC1 was electroporated into O . rhinotracheale and yielded cefoxitin-resistant transformants . The pOREC1 isolated from these transformants was reintroduced into E . coli, demonstrating that pOREC1 acts as an independent replicon in both E . coli and O . rhinotracheale, fulfilling the criteria for a shuttle plasmid that can be used for transformation, targeted mutagenesis, and the construction of defined attenuated vaccine strains . Effects of Lipoprotein Biogenesis Mutations on Flagellar Assembly in Salmonella. Frank E. Dailey, 2002.Flagellar assembly requires the expression of a large number of flagellum-specific genes . However, mutations in a number of other genes in Salmonella and Escherichia coli have been shown to have pleiotropic effects that affect flagellar assembly . FlgH (the L-ring subunit of the flagellar basal body) is a lipoprotein whose modification is important for L-ring assembly . We therefore tested whether the lack of motility of Salmonella mutants defective in lipoprotein biogenesis is a result of inability to modify FlgH . Our results show that temperature-sensitive apolipoprotein N-acyltransferase [lnt(Ts)] mutants are nonflagellate at 42°C . However, the flagellar assembly defect occurs at a much earlier step in the pathway than L-ring assembly . These mutants failed to assemble even an MS ring, presumably because of the observed decrease in transcription of fliF. In contrast, temperature-sensitive diacylglycerol transferase [lgt(Ts)] mutants were motile at 42°C, provided the strains carried an lpp (Braun lipoprotein) mutation to permit growth . We have isolated second-site mutants from an lgt(Ts) lpp+ strain that grow but are nonflagellate at 42°C . Thus, lipoprotein biogenesis is a factor that is important for flagellar assembly . Cytosine Methylation by the SuaI Restriction-Modification System: Implications for Genetic Fidelity in a Hyperthermophilic Archaeon. Dennis W. Grogan, 2003.5-Methylcytosine in chromosomal DNA represents a potential source of frequent spontaneous mutation for hyperthermophiles . To determine the relevance of this threat for the archaeon Sulfolobus acidocaldarius, the mode of GGCC methylation by its restriction-modification system, SuaI, was investigated . Distinct isoschizomers of the SuaI endonuclease were used to probe the methylation state of GGCC in native S . acidocaldarius DNA . In addition, the methylation sensitivity of the SuaI endonuclease was determined with synthetic oligonucleotide substrates and modified natural DNAs . The results show that the SuaI system uses N4 methylation to block cleavage of its recognition site, thereby avoiding the creation of G · T mismatches by spontaneous deamination at extremely high temperature . Heme-Responsive Transcriptional Activation of Bordetella bhu Genes. Carin K. Vanderpool, 2003.Bordetella pertussis and Bordetella bronchiseptica, gram-negative respiratory pathogens of mammals, possess a heme iron utilization system encoded by the bhuRSTUV genes . Preliminary evidence suggested that expression of the BhuR heme receptor was stimulated by the presence of heme under iron-limiting conditions . The hurIR (heme uptake regulator) genes were previously identified upstream of the bhuRSTUV gene cluster and are predicted to encode homologs of members of the iron starvation subfamily of extracytoplasmic function (ECF) regulators . In this study, B . pertussis and B . bronchiseptica  hurI mutants, predicted to lack an ECF
 factor, were constructed and found to be deficient in the utilization of hemin and hemoglobin . Genetic complementation of
hurI strains with plasmid-borne hurI restored wild-type levels of heme utilization . B . bronchiseptica
hurI mutant BRM23 was defective in heme-responsive production of the BhuR heme receptor; hurI in trans restored heme-inducible BhuR expression to the mutant and resulted in BhuR overproduction . Transcriptional analyses with bhuR-lacZ fusion plasmids confirmed that bhuR transcription was activated in iron-starved cells in response to heme compounds . Heme-responsive bhuR transcription was not observed in mutant BRM23, indicating that hurI is required for positive regulation of bhu gene expression . Furthermore, bhuR was required for heme-inducible bhu gene activation, supporting the hypothesis that positive regulation of bhuRSTUV occurs by a surface signaling mechanism involving the heme-iron receptor BhuR .
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