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Reverse Gyrase Is Not a Prerequisite for Hyperthermophilic Life. Haruyuki Atomi, 2004.We disrupted the reverse gyrase gene from a hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1 . An apparent positive supercoiling activity that was observed in the host strain was not found in the disruptant strain . We found that a lack of reverse gyrase led to a retardation in growth that was more striking at higher temperatures . However, the disruption of the reverse gyrase gene did not lead to a lethal phenotype at 90°C . This study provides experimental evidence that reverse gyrase is not a prerequisite for hyperthermophilic life . Membrane Topology of the Multidrug Transporter MdfA: Complementary Gene Fusion Studies Reveal a Nonessential C-Terminal Domain. Julia Adler, 2002.The hydrophobicity profile and sequence alignment of the Escherichia coli multidrug transporter MdfA indicate that it belongs to the 12-transmembrane-domain family of transporters . According to this prediction, MdfA contains a single membrane-embedded charged residue (Glu26), which was shown to play an important role in substrate recognition . To test the predicted secondary structure of MdfA, we analyzed complementary pairs of hybrids of MdfA-PhoA (alkaline phosphatase, functional in the periplasm) and MdfA-Cat (chloramphenicol acetyltransferase, functional in the cytoplasm), generated in all the putative cytoplasmic and periplasmic loops of MdfA . Our results support the 12-transmembrane topology model and the suggestion that except for Glu26, no other charged residues are present in the membrane domain of MdfA . Surprisingly, by testing the ability of the truncated MdfA-Cat and MdfA-PhoA hybrids to confer multidrug resistance, we demonstrate that the entire C-terminal transmembrane domain and the cytoplasmic C terminus are not essential for MdfA-mediated drug resistance and transport . Quorum Sensing Controls Exopolysaccharide Production in Sinorhizobium meliloti. Melanie M. Marketon, 2003.Sinorhizobium meliloti is a soil bacterium capable of invading and establishing a symbiotic relationship with alfalfa plants . This invasion process requires the synthesis, by S . meliloti, of at least one of the two symbiotically important exopolysaccharides, succinoglycan and EPS II . We have previously shown that the sinRI locus of S . meliloti encodes a quorum-sensing system that plays a role in the symbiotic process . Here we show that the sinRI locus exerts one level of control through regulation of EPS II synthesis . Disruption of the autoinducer synthase gene, sinI, abolished EPS II production as well as the expression of several genes in the exp operon that are responsible for EPS II synthesis . This phenotype was complemented by the addition of acyl homoserine lactone (AHL) extracts from the wild-type strain but not from a sinI mutant, indicating that the sinRI-specified AHLs are required for exp gene expression . This was further confirmed by the observation that synthetic palmitoleyl homoserine lactone (C16:1-HL), one of the previously identified sinRI-specified AHLs, specifically restored exp gene expression . Most importantly, the absence of symbiotically active EPS II in a sinI mutant was confirmed in plant nodulation assays, emphasizing the role of quorum sensing in symbiosis . Insertional Mutagenesis of a Fungal Biocontrol Agent Led to Discovery of a Rare Cellobiose Lipid with Antifungal Activity. Yali Cheng, 2003.Insertional mutagenesis was applied for the first time to a fungal biocontrol agent, Pseudozyma flocculosa, in an attempt to obtain mutants with altered antagonistic properties . Transformants were obtained via DNA-mediated transformation . Molecular analyses of the transformants revealed that multiple copies of the plasmid were integrated in tandem at one to many chromosomal loci . The transformants were screened for their biocontrol properties using standard bioassays, and the 160 tested transformants were classified into four groups: group I mutants (22 transformants) showed a stronger antagonistic effect than the wild type (WT) while those of group II (107 transformants) had a comparable antagonistic effect; group III mutants (17 transformants) had a decreased antagonistic effect relative to WT and group IV mutants (14 transformants) had lost their biocontrol properties . Culture extracts of the mutants (group IV) and WT were analyzed and compared for the presence of active metabolites which were then separated by solid-phase extraction and purified using conventional methods . Nuclear magnetic resonance experiments and analytical studies on a metabolite specifically produced by the WT revealed the presence of 2-(2',4'-diacetoxy-5'-carboxy-pentanoyl) octadecyl cellobioside (flocculosin), a novel glycolipid with strong antifungal properties; the production of this compound would account for the biocontrol activity of P . flocculosa .
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