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Novel Archaeal Alanine:Glyoxylate Aminotransferase from Thermococcus litoralis. Haruhiko Sakuraba, 2004.A novel alanine:glyoxylate aminotransferase was found in a hyperthermophilic archaeon, Thermococcus litoralis . The amino acid sequence of the enzyme did not show a similarity to any alanine:glyoxylate aminotransferases reported so far . Homologues of the enzyme appear to be present in almost all hyperthermophilic archaea whose whole genomes have been sequenced . Genetic Structure of a Spodoptera frugiperda Nucleopolyhedrovirus Population: High Prevalence of Deletion Genotypes. Oihane Simón, 2004.A Nicaraguan field isolate (SfNIC) of Spodoptera frugiperda nucleopolyhedrovirus was purified by plaque assay on Sf9 cells . Nine distinct genotypes, A to I, were identified by their restriction endonuclease profiles . Variant SfNIC-B was selected as the standard because its restriction profile corresponded to that of the wild-type isolate . Physical maps were generated for each of the variants . The differences between variants and the SfNIC-B standard were confined to the region between map units 9 and 32.5 . This region included PstI-G, PstI-F, PstI-L, PstI-K and EcoRI-L fragments . Eight genotypes presented a deletion in their genome compared with SfNIC-B . Occlusion body-derived virions of SfNIC-C, -D and -G accounted for 41% of plaque-purified clones . These variants were not infectious per os but retained infectivity by injection into S . frugiperda larvae . Median 50% lethal concentration values for the other cloned genotypes were significantly higher than that of the wild type . The variants also differed in their speed of kill . Noninfectious variants SfNIC-C and -D lacked the pif and pif-2 genes . Infectivity was restored to these variants by plasmid rescue with a plasmid comprising both pif and pif-2 . Transcription of an SfNIC-G gene was detected by reverse transcription-PCR in insects, but no fatal disease developed . Transcription was not detected in SfNIC-C or -D-inoculated larvae . We conclude that the SfNIC population presents high levels of genetic diversity, localized to a 17-kb region containing pif and pif-2, and that interactions among complete and deleted genotypic variants will likely influence the capacity of this virus to control insect pests . Role of OxyR as a Peroxide-Sensing Positive Regulator in Streptomyces coelicolor A3(2). Ji-Sook Hahn, 2002.Genes encoding a homolog of Escherichia coli OxyR (oxyR) and an alkyl hydroperoxide reductase system (ahpC and ahpD) have been isolated from Streptomyces coelicolor A3(2) . The ahpC and ahpD genes constitute an operon transcribed divergently from the oxyR gene. Expression of both ahpCD and oxyR genes was maximal at early exponential phase and decreased rapidly as cells entered mid-exponential phase . Overproduction of OxyR in Streptomyces lividans conferred resistance against cumene hydroperoxide and H2O2 . The oxyR mutant produced fewer ahpCD and oxyR transcripts than the wild type, suggesting that OxyR acts as a positive regulator for their expression . Both oxyR and ahpCD transcripts increased more than fivefold within 10 min of H2O2 treatment and decreased to the normal level in 50 min, with kinetics similar to those of the CatR-mediated induction of the catalase A gene (catA) by H2O2 . The oxyR mutant failed to induce oxyR and ahpCD genes in response to H2O2, indicating that OxyR is the modulator for the H2O2-dependent induction of these genes . Purified OxyR protein bound specifically to the intergenic region between ahpC and oxyR, suggesting its direct role in regulating these genes . These results demonstrate that in S . coelicolor OxyR mediates H2O2 induction of its own gene and genes for alkyl hydroperoxide reductase system, but not the catalase gene (catA), unlike in Escherichia coli and Salmonella enterica serovar Typhimurium . Interactions between the Outer Membrane Ferric Citrate Transporter FecA and TonB: Studies of the FecA TonB Box. Monica Ogierman, 2003.Both induction of transcription of the ferric citrate transport genes and transport of ferric citrate by the Escherichia coli outer membrane receptor FecA require energy derived from the proton motive force (PMF) of the inner membrane . The energy is transduced to FecA by the inner membrane complex, TonB, ExbB, and ExbD . Region 160 of TonB and the conserved TonB box of other TonB-dependent receptors are implicated as sites of interaction . In the present study, the postulated TonB box (D80A81L82T83V84) of FecA was deleted in frame, with a subsequent loss of both FecA functions . DALTV of FecA could be functionally replaced with the core TonB boxes of FhuA (DTITV) and FepA (DTIVV) . Each residue of the TonB box of FecA was sequentially replaced with cysteine residues, and only the D80C replacement showed a loss (reduction) of both FecA functions . A physical interaction between TonB and FecA was demonstrated using both in vivo site-specific disulfide bond cross-linking and nonspecific formaldehyde (FA) cross-linking . Pairwise combinations of FecA (DALTV)/Cys substitutions were cross-linked via disulfide bond formation with TonBQ160C, TonBQ162C, and TonBY163C . Unexpectedly, this cross-linking was not enhanced by substrate (ferric citrate) . In contrast, the TonB-FecA interaction was enhanced by ferric citrate in the FA-cross-linking assay . Energy derived from the PMF was not required for the TonB-FecA interaction in either the disulfide- or FA-cross-linking assay . TonB/CysExbB/ExbD(D25N) was still able to cross-link with the FecA (DALTV)/Cys derivatives in a tonB tolQ background, even though ExbD25N renders the TonB/ExbBD complex nonfunctional (V . Braun, S . Gaisser, C . Herrmann, K . Kampfenkel, H . Killmann, and I . Traub, J . Bacteriol . 178:2836-2845, 1996) . TonB cross-linked to FecA via FA was not inhibited by either carbonylcyanide-m-chlorophenylhydrazone or 1 mM 2,4-dinitrophenol, which dissipate the electrochemical potential of the cytoplasmic membrane and disrupt both FecA functions . The studies shown here demonstrate the significance of the TonB box for FecA functions and are consistent with the view that it is the structure and not the sequence of the TonB box that is important for activity . Demonstrated here for the first time is the physical interaction of TonB and FecA, which is enhanced by ferric citrate . Mode of Bactericidal Action of Silver Zeolite and Its Comparison with That of Silver Nitrate. Yoshinobu Matsumura, 2003.The properties of the bactericidal action of silver zeolite as affected by inorganic salts and ion chelators were similar to those of silver nitrate . The results suggest that the contact of the bacterial cell with silver zeolite, the consequent transfer of silver ion to the cell, and the generation of reactive oxygen species in the cell are involved in the bactericidal activity of silver zeolite .
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