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Thermus thermophilus L11 Methyltransferase, PrmA, Is Dispensable for Growth and Preferentially Modifies Free Ribosomal Protein L11 Prior to Ribosome Assembly. Dale M. Cameron, 2004.The ribosomal protein L11 in bacteria is posttranslationally trimethylated at multiple amino acid positions by the L11 methyltransferase PrmA, the product of the prmA gene . The role of L11 methylation in ribosome function or assembly has yet to be determined, although the deletion of Escherichia coli prmA has no apparent phenotype . We have constructed a mutant of the extreme thermophile Thermus thermophilus in which the prmA gene has been disrupted with the htk gene encoding a heat-stable kanamycin adenyltransferase . This mutant shows no growth defects, indicating that T . thermophilus PrmA, like its E . coli homolog, is dispensable . Ribosomes prepared from this mutant contain unmethylated L11, as determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and are effective substrates for in vitro methylation by cloned and purified T . thermophilus PrmA . MALDI-TOF MS also revealed that T . thermophilus L11 contains a total of 12 methyl groups, in contrast to the 9 methyl groups found in E . coli L11 . Finally, we found that, as with the E . coli methyltransferase, the ribosomal protein L11 dissociated from ribosomes is a more efficient substrate for in vitro methylation by PrmA than intact 70S ribosomes, suggesting that methylation in vivo occurs on free L11 prior to its incorporation into ribosomes . In Vitro Combination of Amdoxovir and the Inosine Monophosphate Dehydrogenase Inhibitors Mycophenolic Acid and Ribavirin Demonstrates Potent Activity against Wild-Type and Drug-Resistant Variants of Human Immunodeficiency Virus Type 1. Katyna Borroto-Esoda, 2004.Amdoxovir [(–)-ß-D-2,6-diaminopurine dioxolane (DAPD)] is a nucleoside analogue reverse transcriptase inhibitor of human immunodeficiency virus type 1 (HIV-1) replication . DAPD is deaminated by adenosine deaminase to the guanosine analogue dioxolane guanosine (DXG), which is subsequently phosphorylated to the corresponding 5' triphosphate (DXG-TP) . DXG-TP competes with the natural substrate dGTP for binding to the enzyme-nucleic acid complex . Mycophenolic acid (MPA) and ribavirin (RBV), inhibitors of inosine monophosphate dehydrogenase (IMPDH), inhibit the de novo synthesis of guanine nucleotides, including dGTP . Reducing the intracellular levels of dGTP would be expected to augment the antiviral activity of analogues of deoxyguanosine . In this study we examined the effect of MPA and RBV on the anti-HIV activity of DAPD and DXG . When tested against wild-type virus, both MPA and RBV decreased the 50% effective concentration (EC50) for DXG by at least 10-fold . In contrast, both MPA and RBV increase the EC50 value for zidovudine . MPA and RBV completely reversed the resistance to DXG observed with HIV isolates containing mutations which confer partial resistance to DAPD and DXG . Similarly, when tested against a mutant virus fully resistant to inhibition by DAPD (K65R/Q151M), MPA and RBV reduced the EC50 for DAPD to within twofold of that for the wild type . The combination of MPA or RBV with DAPD or DXG did not result in increased cytotoxicity or reduced levels of mitochondrial DNA when tested at physiologically relevant concentrations . These studies suggest a potential role for the use of IMPDH inhibitors in combination therapy with amdoxovir in the treatment of HIV . Rubrivivax gelatinosus acsF (Previously orf358) Codes for a Conserved, Putative Binuclear-Iron-Cluster-Containing Protein Involved in Aerobic Oxidative Cyclization of Mg-Protoporphyrin IX Monomethylester. Violaine Pinta, 2002.This study describes the characterization of orf358, an open reading frame of previously unidentified function, in the purple bacterium Rubrivivax gelatinosus . A strain in which orf358 was disrupted exhibited a phenotype similar to the wild type under photosynthesis or low-aeration respiratory growth conditions . In contrast, under highly aerated respiratory growth conditions, the wild type still produced bacteriochlorophyll a (Bchl a), while the disrupted strain accumulated a compound that had the same absorption and fluorescence emission spectra as Mg-protoporphyrin but was less polar, suggesting that it was Mg-protoporphyrin monomethylester (MgPMe) . These data indicated a blockage in Bchl a synthesis at the oxidative cyclization stage and implied the coexistence of two different mechanisms for MgPMe cyclization in R . gelatinosus, an anaerobic mechanism active under photosynthesis or low oxygenation and an aerobic mechanism active under high-oxygenation growth conditions . Based on these results as well as on sequence analysis indicating the presence of conserved putative binuclear-iron-cluster binding motifs, the designation of orf358 as acsF (for aerobic cyclization system Fe-containing subunit) is proposed . Several homologs of AcsF were found in a wide range of photosynthetic organisms, including Chlamydonomas reinhardtii Crd1 and Pharbitis nil PNZIP, suggesting that this aerobic oxidative cyclization mechanism is conserved from bacteria to plants . A 65-Kilobase Pathogenicity Island Is Unique to Philadelphia-1 Strains of Legionella pneumophila. Ann Karen C. Brassinga, 2003.Nucleotide sequence analysis of an  80-kb genomic region revealed an
65-kb locus that bears hallmarks of a pathogenicity island . This locus includes homologues of a type IV secretion system, mobile genetic elements, and known virulence factors . Comparative studies with other Legionella pneumophila strains and serogroups indicated that this
65-kb locus is unique to L . pneumophila serogroup 1 Philadelphia-1 strains .
Molecular Characterization of Natural Erwinia pyrifoliae Strains Deficient in Hypersensitive Response. Susanne Jock, 2003.From necrotic tissue of a Nashi pear tree, 24 Erwinia pyrifoliae strains, found to be identical by pulsed-field gel electrophoresis analysis, were isolated . Thirteen strains were not virulent on immature pears and did not induce a hypersensitive response in tobacco leaves . The defective gene hrpL was complemented with intact genes from E . pyrifoliae and Erwinia amylovora . Density-Dependent Sorting of Physiologically Different Cells of Vibrio parahaemolyticus. Tomohiko Nishino, 2003.A pure bacterial culture is composed of clonal cells in different physiological states . Separation of those subpopulations is critical for further characterization and for understanding various processes in the cultured cells . We used density-dependent cell sorting with Percoll to separate subpopulations from cultures of a marine bacterium, Vibrio parahaemolyticus . Cells from cultures in the exponential and stationary phases were fractionated according to their buoyant density, and their culturability and ability to maintain culturability under low-temperature and low-nutrient stress (stress resistance) were determined . The buoyant density of the major portion of the cells decreased with culture age . The culturability of stationary-phase cells increased with increasing buoyant density, but that of exponential-phase cells did not . Stress resistance decreased with increasing buoyant density regardless of the growth phase . The results indicate that density-dependent cell sorting is useful for separating subpopulations of different culturabilities and stress resistances . We expect that this method will be a powerful tool for analyzing cells in various physiological states, such as the viable but nonculturable state . Multilocus Sequence Typing for Comparison of Veterinary and Human Isolates of Campylobacter jejuni. Georgina Manning, 2003.Multilocus sequence typing (MLST) has been applied to 266 Campylobacter jejuni isolates, mainly from veterinary sources, including cattle, sheep, poultry, pigs, pets, and the environment, as well as isolates from human cases of campylobacteriosis . The populations of veterinary and human isolates overlap, suggesting that most veterinary sources should be considered reservoirs of pathogenic campylobacters . There were some associations between source and sequence type complex, indicating that host or source adaptation may exist . The pig isolates formed a distinct group by MLST and may well represent a potential pig-adapted clone of C . jejuni . A subset (n = 82) of isolates was reanalyzed with a second MLST scheme which provided a unique set of isolates that had been analyzed at a total of 12 loci . The distribution of isolates among the complexes in each of the two schemes was similar but not identical . In addition to isolates from human outbreaks, one group of isolates that were not epidemiologically linked was also identical at all 12 loci . This group of isolates is believed to represent another stable strain of C. jejuni .
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