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RsbV-Independent Induction of the SigB-Dependent General Stress Regulon of Bacillus subtilis during Growth at High Temperature. Gudrun Holtmann, 2004.General stress proteins protect Bacillus subtilis cells against a variety of environmental insults . This adaptive response is particularly important for nongrowing cells, to which it confersa multiple, nonspecific, and preemptive stress resistance . Inductionof the general stress response relies on the alternative transcription factor, SigB, whose activity is controlled by a partner switching mechanism that also involves the anti-sigma factor, RsbW, andthe antagonist protein, RsbV . Recently, the SigB regulon hasbeen shown to be continuously induced and functionally importantin cells actively growing at low temperature . With the exceptionof this chill induction, all SigB-activating stimuli identifiedso far trigger a transient expression of the SigB regulon thatdepends on RsbV . Through a proteome analysis and Northern blotand gene fusion experiments, we now show that the SigB regulonis continuously induced in cells growing actively at 51°C,close to the upper growth limit of B . subtilis . This heat inductionof SigB-dependent genes requires the environmental stress-responsivephosphatase RsbU, but not the metabolic stress-responsive phosphataseRsbP . RsbU dependence of SigB activation by heat is overcomein mutants that lack RsbV . In addition, loss of RsbV alone orin combination with RsbU triggers a hyperactivation of the generalstress regulon exclusively at high temperatures detrimentalfor cell growth . These new facets of heat induction of the SigBregulon indicate that the current view of the complex geneticand biochemical regulation of SigB activity is still incompleteand that SigB perceives signals independent of the RsbV-mediatedsignal transduction pathways under heat stress conditions. Use of the Riboflavin Synthase Gene (ribC) as a Model for Development of an Essential Gene Disruption and Complementation System for Haemophilus influenzae. Amna Saeed-Kothe, 2004.We have developed a system for rapid and reliable assessment of gene essentiality in Haemophilus influenzae Rd strain KW20 . We constructed two "suicide" complementation vectors (pASK5 and pASK6) containing 5' and 3' regions of the nonessential ompP1 gene flanking a multiple cloning site and a selectable marker (a chloramphenicol resistance gene or a tetracycline resistance cassette) . Transformation of H . influenzae with the complementation constructs directs chromosomal integration of a gene of interest into the ompP1 locus, where the strong, constitutive ompP1 promoter drives its expression . This single-copy, chromosome-based complementation system is useful for confirming the essentiality of disrupted genes of interest . It allows genetic analysis in a background free of interference from any upstream or downstream genetic elements and enables conclusive assignment of essentiality . We validated this system by using the riboflavin synthase gene (ribC), a component of the riboflavin biosynthetic pathway . Our results confirmed the essentiality of ribC for survival of H . influenzae Rd strain KW20 and demonstrated that a complementing copy of ribC placed under control of the ompP1 promoter reverses the lethal phenotype of a strain with ribC deleted . Physiological Responses of the Hyperthermophilic Archaeon "Pyrococcus abyssi" to DNA Damage Caused by Ionizing Radiation. Edmond Jolivet, 2003.The mechanisms by which hyperthermophilic Archaea, such as "Pyrococcus abyssi" and Pyrococcus furiosus, survive high doses of ionizing gamma irradiation are not thoroughly elucidated . Following gamma-ray irradiation at 2,500 Gy, the restoration of "P . abyssi" chromosomes took place within chromosome fragmentation . DNA synthesis in irradiated "P . abyssi" cells during the DNA repair phase was inhibited in comparison to nonirradiated control cultures, suggesting that DNA damage causes a replication block in this organism . We also found evidence for transient export of damaged DNA out of irradiated "P . abyssi" cells prior to a restart of chromosomal DNA synthesis . Our cell fractionation assays further suggest that "P . abyssi" contains a highly efficient DNA repair system which is continuously ready to repair the DNA damage caused by high temperature and/or ionizing radiation . Thermostabilization of Bacterial Fructosyl-Amino Acid Oxidase by Directed Evolution. Ryoichi Sakaue, 2003.We succeeded in isolating several thermostable mutant fructosyl-amino acid oxidase (FAOX; EC 1.5.3) without reduction of productivity by directed evolution that combined an in vivo mutagenesis and membrane assay screening system . Five amino acid substitutions (T60A, A188G, M244L, N257S, and L261M) occurred in the most thermostable mutant obtained by a fourth round of directed evolution . This altered enzyme, FAOX-TE, was stable at 45°C, whereas the wild-type enzyme was not stable above 37°C . The Km values of FAOX-TE for D-fructosyl-L-valine and D-fructosyl-glycine were 1.50 and 0.58 mM, respectively, in contrast with corresponding values of 1.61 and 0.74 mM for the wild-type enzyme . This altered FAOX-TE will be useful in the diagnosis of diabetes .
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