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Correspondence between Community Structure and Function during Succession in Phenol- and Phenol-plus-Trichloroethene-Fed Sequencing Batch Reactors. Héctor L. Ayala-del-Río, 2004.The effects of more than 2 years of trichloroethene (TCE) application on community succession and function were studied in two aerobic sequencing batch reactors . One reactor was fed phenol, and the second reactor was fed both phenol and TCE in sequence twice per day . After initiation of TCE loading in the second reactor, the TCE transformation rates initially decreased, but they stabilized with an average second-order rate coefficient of 0.044 liter mg–1 day–1 for 2 years . In contrast, the phenol-fed reactor showed higher and unstable TCE transformation rates, with an average rate coefficient of 0.093 liter mg–1 day–1 . Community analysis by terminal restriction fragment length polymorphism (T-RFLP) analysis of the 16S rRNA genes showed that the phenol-plus-TCE-fed reactor had marked changes in community structure during the first 100 days and remained relatively stable afterwards, corresponding to the period of stable function . In contrast, the community structure of the phenol-fed reactor changed periodically, and the changes coincided with the periodicity observed in the TCE transformation rates . Correspondence analysis of each reactor community showed that different community structures corresponded with function (TCE degradation rate) . Furthermore, the phenol hydroxylase genotypes, as determined by restriction fragment length polymorphism analysis, corresponded to community structure patterns identified by T-RFLP analysis and to periods when the TCE transformation rates were high . Long-term TCE stress appeared to select for a different and stable community structure, with lower but stable TCE degradation rates . In contrast, the community under no stress exhibited a dynamic structure and dynamic function . In Vivo Assay for Low-Activity Mutant Forms of Escherichia coli Ribonucleotide Reductase. Monica Ekberg, 2003.Ribonucleotide reductase (RNR) catalyzes the essential production of deoxyribonucleotides in all living cells . In this study we have established a sensitive in vivo assay to study the activity of RNR in aerobic Escherichia coli cells . The method is based on the complementation of a chromosomally encoded nonfunctional RNR with plasmid-encoded RNR . This assay can be used to determine in vivo activity of RNR mutants with activities beyond the detection limits of traditional in vitro assays . E . coli RNR is composed of two homodimeric proteins, R1 and R2 . The R2 protein contains a stable tyrosyl radical essential for the catalysis that takes place at the R1 active site . The three-dimensional structures of both proteins, phylogenetic studies, and site-directed mutagenesis experiments show that the radical is transferred from the R2 protein to the active site in the R1 protein via a radical transfer pathway composed of at least nine conserved amino acid residues . Using the new assay we determined the in vivo activity of mutants affecting the radical transfer pathway in RNR and identified some residual radical transfer activity in two mutant R2 constructs (D237N and W48Y) that had previously been classified as negative for enzyme activity . In addition, we show that the R2 mutant Y356W is completely inactive, in sharp contrast to what has previously been observed for the corresponding mutation in the mouse R2 enzyme .
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