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Polar Localization of Replicon Origins in the Multipartite Genomes of Agrobacterium tumefaciens and Sinorhizobium meliloti. Lyn Sue Kahng, 2003.The origins of replication of many different bacteria have been shown to reside at specific subcellular locations, but the mechanisms underlying their positioning and segregation are still being elucidated . In particular, little is known about the replication of multipartite genomes in bacteria . We determined the cellular positions of the origins of the replicons in the alpha proteobacteria Agrobacterium tumefaciens and Sinorhizobium meliloti and found that they are located at the poles of the cells . Our work demonstrates the conserved extreme polar localization of circular chromosome origins in these alpha proteobacteria and is also the first to specify the cellular location of origin regions from the repABC family . The cellular location of a derivative of the RK2 plasmid is distinct from that of the alpha proteobacterium genomic replicon origins but is conserved across bacteria . Colocalization experiments with the genomic replicons of A . tumefaciens revealed that the repABC replicons, although preferentially positioned at the cell pole, colocalize only rarely . For the repABC replicons in this organism, occupying discrete spatial locations may contribute to their coexistence and stable inheritance . Modified Fixed-Ratio Isobologram Method for Studying In Vitro Interactions between Atovaquone and Proguanil or Dihydroartemisinin against Drug-Resistant Strains of Plasmodium falciparum. Quinton L. Fivelman, 2004.A modified fixed-ratio isobologram method for studying the in vitro interactions between antiplasmodial drugs is described . This method was used to examine the interactions between atovaquone, proguanil, and dihydroartemisinin . The interaction between atovaquone and proguanil was synergistic against atovaquone-sensitive strains K1 and T996; however, there was a loss of synergy against atovaquone-resistant strain NGATV01 isolated after Malarone (the combination of atovaquone and proguanil) treatment failure . While the interaction between atovaquone and dihydroartemisinin was indifferent against isolate NGATV01, the interaction displayed indifference tending toward antagonism against the atovaquone-sensitive strains tested . The relevance of in vitro interactions to in vivo treatment is discussed . Membrane Binding by MinD Involves Insertion of Hydrophobic Residues within the C-Terminal Amphipathic Helix into the Bilayer. Huaijin Zhou, 2003.MinD binds to phospholipid vesicles in the presence of ATP and is released by MinE, which stimulates the MinD ATPase . Membrane binding requires a short conserved C-terminal region, which has the potential to form an amphipathic helix . This finding has led to a model in which the binding of ATP regulates the formation or accessibility of this helix, which then embeds in the membrane bilayer . To test this model, we replaced each of the four hydrophobic residues within this potential helix with tryptophan or a charged residue . Introduction of a negatively charged amino acid decreased membrane binding of MinD and its ability to activate MinC . In contrast, mutants with tryptophan substitutions retained the ability to bind to the membrane and activate MinC . Fluorescence emission spectroscopy analysis of the tryptophan mutants F263W, L264W, and L267W confirmed that these tryptophan residues did insert into the hydrophobic interior of the bilayer . We conclude that membrane binding by MinD involves penetration of the hydrophobic residues within the C-terminal amphipathic helix into the hydrophobic interior of the bilayer . Phylogenetic Diversity of Lactic Acid Bacteria Associated with Paddy Rice Silage as Determined by 16S Ribosomal DNA Analysis. Saïd Ennahar, 2003.A total of 161 low-G+C-content gram-positive bacteria isolated from whole-crop paddy rice silage were classified and subjected to phenotypic and genetic analyses . Based on morphological and biochemical characters, these presumptive lactic acid bacterium (LAB) isolates were divided into 10 groups that included members of the genera Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, and Weissella . Analysis of the 16S ribosomal DNA (rDNA) was used to confirm the presence of the predominant groups indicated by phenotypic analysis and to determine the phylogenetic affiliation of representative strains . The virtually complete 16S rRNA gene was PCR amplified and sequenced . The sequences from the various LAB isolates showed high degrees of similarity to those of the GenBank reference strains (between 98.7 and 99.8%) . Phylogenetic trees based on the 16S rDNA sequence displayed high consistency, with nodes supported by high bootstrap values . With the exception of one species, the genetic data was in agreement with the phenotypic identification . The prevalent LAB, predominantly homofermentative (66%), consisted of Lactobacillus plantarum (24%), Lactococcus lactis (22%), Leuconostoc pseudomesenteroides (20%), Pediococcus acidilactici (11%), Lactobacillus brevis (11%), Enterococcus faecalis (7%), Weissella kimchii (3%), and Pediococcus pentosaceus (2%) . The present study, the first to fully document rice-associated LAB, showed a very diverse community of LAB with a relatively high number of species involved in the fermentation process of paddy rice silage . The comprehensive 16S rDNA-based approach to describing LAB community structure was valuable in revealing the large diversity of bacteria inhabiting paddy rice silage and enabling the future design of appropriate inoculants aimed at improving its fermentation quality . Influence of Nutritional Factors on the Nature, Yield, and Composition of Exopolysaccharides Produced by Gluconacetobacter xylinus I-2281. Henri Kornmann, 2003.The influence of substrate composition on the yield, nature, and composition of exopolysaccharides (EPS) produced by the food-grade strain Gluconacetobacter xylinus I-2281 was investigated during controlled cultivations on mixed substrates containing acetate and either glucose, sucrose, or fructose . Enzymatic activity analysis and acid hydrolysis revealed that two EPS, gluconacetan and levan, were produced by G . xylinus. In contrast to other acetic acid strains, no exocellulose formation has been measured . Considerable differences in metabolite yields have been observed with regard to the carbohydrate source . It was shown that glucose was inadequate for EPS production since most of this substrate (0.84 C-mol/C-mol) was oxidized into gluconic acid, 2-ketogluconic acid, and 5-ketogluconic acid . In contrast, sucrose and fructose supported a 0.35 C-mol/C-mol gluconacetan yield . In addition, growing G . xylinus on sucrose produced a 0.07 C-mol/C-mol levan yield . The composition of EPS remained unchanged during the course of the fermentations . Levan sucrase activity was found to be mainly membrane associated . In addition to levan production, an analysis of levan sucrase's activity also explained the formation of glucose oxides during fermentation on sucrose through the release of glucose . The biosynthetic pathway of gluconacetan synthesis has also been explored . Although the activity of key enzymes showed large differences to be a function of the carbon source, the ratio of their activities remained similar from one carbon source to another and corresponded to the ratio of precursor needs as deduced from the gluconacetan composition .
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